Reduction of flake-like aggregation in nanoparticulate active agent compositions

ABSTRACT

This invention is directed to reduction of flake-like aggregation in nanoparticulate compositions. Also encompassed by the invention are compositions comprising a nanoparticulate active agent, at least one surface stabilizer and a flake-like aggregation reducing agent, such as a buffer and a sugar. The nanoparticulate active agent compositions comprise particles of the active agent having an effective average particle size of less than about 2000 nm.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/130,255, filed Apr. 15, 2016, now U.S. Pat. No. 9,974,746, which is acontinuation of U.S. patent application Ser. No. 14/133,191, filed Dec.18, 2013, now U.S. Pat. No. 9,345,665, which is a divisional of U.S.patent application Ser. No. 12/788,196, filed May 26, 2010, which claimspriority to U.S. Provisional Patent Application No. 61/181,641, filedMay 27, 2009. The contents of these applications are incorporated hereinby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to reduction of flake-likeaggregation in nanoparticulate active agent compositions. Morespecifically, the invention relates to compositions comprising ananoparticulate active agent, at least one surface stabilizer and aflake-like aggregation reducing agent. The nanoparticulate active agentcompositions comprise particles of an active agent having an effectiveaverage particle size of less than about 2000 nm.

BACKGROUND OF THE INVENTION

A. Background Regarding Nanoparticulate Active Agent Compositions

Nanoparticulate active agent compositions, first described in U.S. Pat.No. 5,145,684 (“the '684 patent”), are particles comprising a poorlysoluble therapeutic or diagnostic agent having adsorbed onto orassociated with the surface thereof a non-crosslinked surfacestabilizer.

Methods of making nanoparticulate active agent compositions aredescribed in, for example, U.S. Pat. Nos. 5,518,187 and 5,862,999, bothfor “Method of Grinding Pharmaceutical Substances;” U.S. Pat. No.5,718,388, for “Continuous Method of Grinding PharmaceuticalSubstances;” and U.S. Pat. No. 5,510,118 for “Process of PreparingTherapeutic Compositions Containing Nanoparticles.”

Nanoparticulate active agent compositions are also described, forexample, in U.S. Pat. Nos. 5,298,262 for “Use of Ionic Cloud PointModifiers to Prevent Particle Aggregation During Sterilization;”5,302,401 for “Method to Reduce Particle Size Growth DuringLyophilization;” 5,318,767 for “X-Ray Contrast Compositions Useful inMedical Imaging;” 5,326,552 for “Novel Formulation For NanoparticulateX-Ray Blood Pool Contrast Agents Using High Molecular Weight Non-ionicSurfactants;” 5,328,404 for “Method of X-Ray Imaging Using IodinatedAromatic Propanedioates;” 5,336,507 for “Use of Charged Phospholipids toReduce Nanoparticle Aggregation;” 5,340,564 for “Formulations ComprisingOlin 10-G to Prevent Particle Aggregation and Increase Stability;”5,346,702 for “Use of Non-Ionic Cloud Point Modifiers to MinimizeNanoparticulate Aggregation During Sterilization;” 5,349,957 for“Preparation and Magnetic Properties of Very Small Magnetic-DextranParticles;” 5,352,459 for “Use of Purified Surface Modifiers to PreventParticle Aggregation During Sterilization;” 5,399,363 and 5,494,683,both for “Surface Modified Anticancer Nanoparticles;” 5,401,492 for“Water Insoluble Non-Magnetic Manganese Particles as Magnetic ResonanceEnhancement Agents;” 5,429,824 for “Use of Tyloxapol as aNanoparticulate Stabilizer;” 5,447,710 for “Method for MakingNanoparticulate X-Ray Blood Pool Contrast Agents Using High MolecularWeight Non-ionic Surfactants;” 5,451,393 for “X-Ray ContrastCompositions Useful in Medical Imaging;” 5,466,440 for “Formulations ofOral Gastrointestinal Diagnostic X-Ray Contrast Agents in Combinationwith Pharmaceutically Acceptable Clays;” 5,470,583 for “Method ofPreparing Nanoparticle Compositions Containing Charged Phospholipids toReduce Aggregation;” 5,472,683 for “Nanoparticulate Diagnostic MixedCarbamic Anhydrides as X-Ray Contrast Agents for Blood Pool andLymphatic System Imaging;” 5,500,204 for “Nanoparticulate DiagnosticDimers as X-Ray Contrast Agents for Blood Pool and Lymphatic SystemImaging;” 5,518,738 for “Nanoparticulate NSAID Formulations;” 5,521,218for “Nanoparticulate Iododipamide Derivatives for Use as X-Ray ContrastAgents;” 5,525,328 for “Nanoparticulate Diagnostic Diatrizoxy EsterX-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;”5,543,133 for “Process of Preparing X-Ray Contrast CompositionsContaining Nanoparticles;” 5,552,160 for “Surface Modified NSAIDNanoparticles;” 5,560,931 for “Formulations of Compounds asNanoparticulate Dispersions in Digestible Oils or Fatty Acids;”5,565,188 for “Polyalkylene Block Copolymers as Surface Modifiers forNanoparticles;” 5,569,448 for “Sulfated Non-ionic Block CopolymerSurfactant as Stabilizer Coatings for Nanoparticle Compositions;”5,571,536 for “Formulations of Compounds as Nanoparticulate Dispersionsin Digestible Oils or Fatty Acids;” 5,573,749 for “NanoparticulateDiagnostic Mixed Carboxylic Anydrides as X-Ray Contrast Agents for BloodPool and Lymphatic System Imaging;” 5,573,750 for “Diagnostic ImagingX-Ray Contrast Agents;” 5,573,783 for “Redispersible NanoparticulateFilm Matrices With Protective Overcoats;” 5,580,579 for “Site-specificAdhesion Within the GI Tract Using Nanoparticles Stabilized by HighMolecular Weight, Linear Poly(ethylene Oxide) Polymers;” 5,585,108 for“Formulations of Oral Gastrointestinal Therapeutic Agents in Combinationwith Pharmaceutically Acceptable Clays;” 5,587,143 for “ButyleneOxide-Ethylene Oxide Block Copolymers Surfactants as Stabilizer Coatingsfor Nanoparticulate Compositions;” 5,591,456 for “Milled Naproxen withHydroxypropyl Cellulose as Dispersion Stabilizer;” 5,593,657 for “NovelBarium Salt Formulations Stabilized by Non-ionic and AnionicStabilizers;” 5,622,938 for “Sugar Based Surfactant for Nanocrystals;”5,628,981 for “Improved Formulations of Oral Gastrointestinal DiagnosticX-Ray Contrast Agents and Oral Gastrointestinal Therapeutic Agents;”5,643,552 for “Nanoparticulate Diagnostic Mixed Carbonic Anhydrides asX-Ray Contrast Agents for Blood Pool and Lymphatic System Imaging;”5,718,388 for “Continuous Method of Grinding Pharmaceutical Substances;”5,718,919 for “Nanoparticles Containing the R(−) Enantiomer ofIbuprofen;” 5,747,001 for “Aerosols Containing BeclomethasoneNanoparticle Dispersions;” 5,834,025 for “Reduction of IntravenouslyAdministered Nanoparticulate Formulation Induced Adverse PhysiologicalReactions;” 6,045,829 “Nanocrystalline Formulations of HumanImmunodeficiency Virus (HIV) Protease Inhibitors Using CellulosicSurface Stabilizers;” 6,068,858 for “Methods of Making NanocrystallineFormulations of Human Immunodeficiency Virus (HIV) Protease InhibitorsUsing Cellulosic Surface Stabilizers;” 6,153,225 for “InjectableFormulations of Nanoparticulate Naproxen;” 6,165,506 for “New Solid DoseForm of Nanoparticulate Naproxen;” 6,221,400 for “Methods of TreatingMammals Using Nanocrystalline Formulations of Human ImmunodeficiencyVirus (HIV) Protease Inhibitors;” 6,264,922 for “Nebulized AerosolsContaining Nanoparticle Dispersions;” 6,267,989 for “Methods forPreventing Crystal Growth and Particle Aggregation in NanoparticleCompositions;” 6,270,806 for “Use of PEG-Derivatized Lipids as SurfaceStabilizers for Nanoparticulate Compositions;” 6,316,029 for “RapidlyDisintegrating Solid Oral Dosage Form,” 6,375,986 for “Solid DoseNanoparticulate Compositions Comprising a Synergistic Combination of aPolymeric Surface Stabilizer and Dioctyl Sodium Sulfosuccinate;”6,428,814 for “Bioadhesive Nanoparticulate Compositions Having CationicSurface Stabilizers;” 6,431,478 for “Small Scale Mill;” 6,432,381 for“Methods for Targeting Drug Delivery to the Upper and/or LowerGastrointestinal Tract,” 6,592,903 for “Nanoparticulate DispersionsComprising a Synergistic Combination of a Polymeric Surface Stabilizerand Dioctyl Sodium Sulfosuccinate,” 6,582,285 for “Apparatus forsanitary wet milling;” 6,656,504 for “Nanoparticulate CompositionsComprising Amorphous Cyclosporine;” 6,742,734 for “System and Method forMilling Materials;” 6,745,962 for “Small Scale Mill and Method Thereof;”6,811,767 for “Liquid droplet aerosols of nanoparticulate drugs;”6,908,626 for “Compositions having a combination of immediate releaseand controlled release characteristics;” 6,969,529 for “Nanoparticulatecompositions comprising copolymers of vinyl pyrrolidone and vinylacetate as surface stabilizers;” and 6,976,647 for “System and Methodfor Milling Materials,” 6,991,191 for “Method of Using a Small ScaleMill;” 7,101,576 for “Nanoparticulate Megestrol Formulation,” 7,198,795for “In vitro methods for evaluating the in vivo effectiveness of dosageforms of microparticulate of nanoparticulate active agent compositions;”7,244,451 for “Methods of making nanoparticulate drug compositionscomprising copolymers of vinyl pyrrolidone and vinyl acetate as surfacestabilizers”; 7,276,249 for “Nanoparticulate Fibrate Formulations”;7,288,267 for “Bioadhesive nanoparticulate compositions having cationicsurface stabilizers”; 7,320,802 for “Methods of treatment usingnanoparticulate fenofibrate compositions”; and 7,390,505 for“Nanoparticulate topiramate formulations”, all of which are specificallyincorporated by reference.

Nanoparticulate active agent compositions are also described in U.S.Patent Publication No. 20080152720 for “Nanoparticulate tacrolimusformulations”; U.S. Patent Publication No. 20080152585 for “Lowviscosity liquid dosage forms”; U.S. Patent Publication No. 20080138424for “Nanoparticulate fibrate formulations”; U.S. Patent Publication No.20080124393 for “Controlled release nanoparticulate compositions”; U.S.Patent Publication No. 20080124389 for “Nanoparticulate and ControlledRelease Compositions Comprising Cyclosporine”; U.S. Patent PublicationNo. 20080113025 for “Compositions Comprising Nanoparticulate Naproxenand Controlled Release Hydrocodone”; U.S. Patent Publication No.20080107741 for “Nanoparticulate Compositions of AngiogenesisInhibitors”; U.S. Patent Publication No. 20080102121 for “CompositionsComprising Nanoparticulate Meloxicam and Controlled ReleaseHydrocodone”; U.S. Patent Publication No. 20080095851 for“Nanoparticulate fibrate formulations”; U.S. Patent Publication No.20080050461 for “Nanoparticulate Compositions of AngiogenesisInhibitors”; U.S. Patent Publication No. 20080025807 for “System andMethod for Milling Materials”; U.S. Patent Publication No. 20080003295for “Bioadhesive nanoparticulate compositions having cationic surfacestabilizers”; U.S. Patent Publication No. 20070298115 for“Nanoparticulate fibrate formulations”; U.S. Patent Publication No.20070298098 for “Controlled Release Compositions ComprisingLevetiracetam”; U.S. Patent Publication No. 20070281011 for“Nanoparticulate posaconazole formulations”; U.S. Patent Publication No.20070264348 for “Nanoparticulate fibrate formulations”; U.S. PatentPublication No. 20070224279 for “Stabilization of chemical compoundsusing nanoparticulate formulations”; U.S. Patent Publication No.20070202180 for “Nanoparticulate carvedilol formulations”; U.S. PatentPublication No. 20070178051 for “Sterilized NanoparticulateGlucocorticosteroid Formulations”; U.S. Patent Publication No.20070160675 for “Nanoparticulate and controlled release compositionscomprising a cephalosporin”; U.S. Patent Publication No. 20070148100 for“Nanoparticulate aripiprazole formulations”; U.S. Patent Publication No.20070141159 for “Methods of Making Nanoparticulate CompositionsComprising Copolymers of Vinyl Pyrrolidone and Vinyl Acetate as SurfaceStabilizers”; U.S. Patent Publication No. 20070134339 for “Zonisamideand NSAID Nanoparticulate Formulations”; U.S. Patent Publication No.20070122486 for “Nanoparticulate insulin”; U.S. Patent Publication No.20070110776 for “In vitro methods for evaluating the in vivoeffectiveness of dosage forms of microparticulate or nanoparticulateactive agent compositions;” U.S. Patent Publication No. 20070104792 for“Nanoparticulate tadalafil formulations;” U.S. Patent Publication No.20070098805 for “Methods of making and using novel griseofulvincompositions;” U.S. Patent Publication No. 20070065374 for“Nanoparticulate leukotriene receptor antagonist/corticosteroidformulations;” U.S. Patent Publication No. 20070059371 for“Nanoparticulate ebastine formulations;” U.S. Patent Publication No.20070048378 for “Nanoparticulate anticonvulsant and immunosuppressivecompositions;” U.S. Patent Publication No. 20070042049 for“Nanoparticulate benidipine compositions;” U.S. Patent Publication No.20070015719 for “Nanoparticulate clarithromycin formulations;” U.S.Patent Publication No. 20070003628 for “Nanoparticulate clopidogrelformulations;” U.S. Patent Publication No. 20070003615 for“Nanoparticulate clopidogrel and aspirin combination formulations;” U.S.Patent Publication No. 20060292214 for “Nanoparticulate acetaminophenformulations;” U.S. Patent Publication No. 20060275372 for“Nanoparticulate imatinib mesylate formulations;” U.S. PatentPublication No. 20060246142 for “Nanoparticulate quinazoline derivativeformulations,” U.S. Patent Publication No. 20060246141 for“Nanoparticulate lipase inhibitor formulations,” U.S. Patent PublicationNo. 20060216353 for “Nanoparticulate corticosteroid and antihistamineformulations,” U.S. Patent Publication No. 20060210639 for“Nanoparticulate bisphosphonate compositions,” U.S. Patent PublicationNo. 20060210638 for “Injectable compositions of nanoparticulateimmunosuppressive compounds,” U.S. Patent Publication No. 20060204588for “Formulations of a nanoparticulate finasteride, dutasteride ortamsulosin hydrochloride, and mixtures thereof,” U.S. Patent PublicationNo. 20060198896 for “Aerosol and injectable formulations ofnanoparticulate benzodiazepine,” U.S. Patent Publication No. 20060193920for “Nanoparticulate Compositions of Mitogen-Activated (MAP) KinaseInhibitors,” U.S. Patent Publication No. 20060188566 for“Nanoparticulate formulations of docetaxel and analogues thereof,” U.S.Patent Publication No. 20060165806 for “Nanoparticulate candesartanformulations,” U.S. Patent Publication No. 20060159767 for“Nanoparticulate bicalutamide formulations,” U.S. Patent Publication No.20060159766 for “Nanoparticulate tacrolimus formulations,” U.S. PatentPublication No. 20060159628 for “Nanoparticulate benzothiopheneformulations,” U.S. Patent Publication No. 20060154918 for “Injectablenanoparticulate olanzapine formulations,” U.S. Patent Publication No.20060121112 for “Topiramate pharmaceutical composition,” U.S. PatentPublication No. 20020012675 A1, for “Controlled Release NanoparticulateCompositions,” U.S. Patent Publication No. 20040195413 A1, for“Compositions and method for milling materials,” U.S. Patent PublicationNo. 20040173696 A1 for “Milling microgram quantities of nanoparticulatecandidate compounds,” U.S. Patent Application No. 20020012675 A1,published on Jan. 31, 2002, for “Controlled Release NanoparticulateCompositions;” U.S. Patent Publication No. 20050276974 for“Nanoparticulate Fibrate Formulations;” U.S. Patent Publication No.20050238725 for “Nanoparticulate compositions having a peptide as asurface stabilizer;” U.S. Patent Publication No. 20050233001 for“Nanoparticulate megestrol formulations;” U.S. Patent Publication No.20050147664 for “Compositions comprising antibodies and methods of usingthe same for targeting nanoparticulate active agent delivery;” U.S.Patent Publication No. 20050063913 for “Novel metaxalone compositions;”U.S. Patent Publication No. 20050042177 for “Novel compositions ofsildenafil free base;” U.S. Patent Publication No. 20050031691 for “Gelstabilized nanoparticulate active agent compositions;” U.S. PatentPublication No. 20050019412 for “ Novel glipizide compositions;” U.S.Patent Publication No. 20050004049 for “Novel griseofulvincompositions;” U.S. Patent Publication No. 20040258758 for“Nanoparticulate topiramate formulations;” U.S. Patent Publication No.20040258757 for “Liquid dosage compositions of stable nanoparticulateactive agents;” U.S. Patent Publication No. 20040229038 for“Nanoparticulate meloxicam formulations;” U.S. Patent Publication No.20040208833 for “Novel fluticasone formulations;” U.S. PatentPublication No. 20040195413 for “ Compositions and method for millingmaterials;” U.S. Patent Publication No. 20040156895 for “Solid dosageforms comprising pullulan;” U.S. Patent Publication No. U.S. PatentPublication No. U.S. Patent Publication No. 20040156872 for “Novelnimesulide compositions;” U.S. Patent Publication No. 20040141925 for“Novel triamcinolone compositions;” U.S. Patent Publication No.20040115134 for “Novel nifedipine compositions;” U.S. Patent PublicationNo. 20040105889 for “Low viscosity liquid dosage forms;” U.S. PatentPublication No. 20040105778 for “Gamma irradiation of solidnanoparticulate active agents;” U.S. Patent Publication No. 20040101566for “Novel benzoyl peroxide compositions;” U.S. Patent Publication No.20040057905 for “Nanoparticulate beclomethasone dipropionatecompositions;” U.S. Patent Publication No. 20040033267 for“Nanoparticulate compositions of angiogenesis inhibitors;” U.S. PatentPublication No. 20040033202 for “Nanoparticulate sterol formulations andnovel sterol combinations;” U.S. Patent Publication No. 20040018242 for“Nanoparticulate nystatin formulations;” U.S. Patent Publication No.20040015134 for “Drug delivery systems and methods;” U.S. PatentPublication No. 20030232796 for “Nanoparticulate polycosanolformulations & novel polycosanol combinations;” U.S. Patent PublicationNo. 20030215502 for “Fast dissolving dosage forms having reducedfriability;” U.S. Patent Publication No. 20030185869 for“Nanoparticulate compositions having lysozyme as a surface stabilizer;”U.S. Patent Publication No. 20030181411 for “Nanoparticulatecompositions of mitogen-activated protein (MAP) kinase inhibitors;” U.S.Patent Publication No. 20030137067 for “Compositions having acombination of immediate release and controlled releasecharacteristics;” U.S. Patent Publication No. 20030108616 for“Nanoparticulate compositions comprising copolymers of vinyl pyrrolidoneand vinyl acetate as surface stabilizers;” U.S. Patent Publication No.20030095928 for “Nanoparticulate insulin;” U.S. Patent Publication No.20030087308 for “Method for high through put screening using a smallscale mill or microfluidics;” U.S. Patent Publication No. 20030023203for “Drug delivery systems & methods;” U.S. Patent Publication No.20020179758 for “System and method for milling materials; and U.S.Patent Publication No. 20010053664 for “Apparatus for sanitary wetmilling,” describe nanoparticulate active agent compositions and arespecifically incorporated by reference.

Amorphous small particle compositions are described, for example, inU.S. Pat. Nos. 4,783,484 for “Particulate Composition and Use Thereof asAntimicrobial Agent;” 4,826,689 for “Method for Making Uniformly SizedParticles from Water-Insoluble Organic Compounds;” 4,997,454 for “Methodfor Making Uniformly-Sized Particles From Insoluble Compounds;”5,741,522 for “Ultrasmall, Non-aggregated Porous Particles of UniformSize for Entrapping Gas Bubbles Within and Methods;” and 5,776,496, for“Ultrasmall Porous Particles for Enhancing Ultrasound Back Scatter.”These disclosures are also specifically incorporated by reference.

None of these references describe the phenomenon of reducing“flake-like” aggregation in a nanoparticulate composition or a mechanismof reducing the same.

B. Background Regarding Injectable Formulations

The skilled person knows that for any particulate composition to beapproved by the FDA for intravenous (I.V.) or intramuscular (I.M.)administration, the composition must meet the standards set forth inGeneral Chapter 788 of the United States Pharmacopeia (“USP<788>”).Specifically, in the United States, any particulate matter injectablesolution must comply with the particle size and number requirements ofUSP<788>. That is, under the approved “Light Obscuration” test set forthin USP<788>, known as “Method 1,” there must be: (i) no more than 6,000particles in a particulate composition that are greater than 10 μm insize and (ii) no more than 600 particles that are greater than 25 μm insize. Under “Method 2,” the Microscopy test, a particulate compositionmust contain (i) no more than 3,000 particles in a particulatecomposition that are greater than 10 μm in size and (ii) no more than300 particles that are greater than 25 μm in size. The theorized largeparticles represent the presence of aggregates of individual particleswhich clump together. It is theorized that nano particulate formulationshas an inherent propensity to form flake like aggregates (“FLA”),presumably caused by colloidal surface phenomena. The “FLA's” arepresumably formed at the air-liquid interface as the aggregatemorphology is extremely two dimensional in its overall shape. Suchaggregates have commonly been observed using optical microscopy andscanning electron microscopy techniques, but have not been detectedusing light scattering based particle sizing techniques.

There is a need in the art to develop injectable nanoparticulate activeagent formulations that are essentially free of “flake-like”particulates, that meet the USP<788> criteria for particulate mater, andthat have better stability at room temperature. Ideally, theformulations are ready-for-use, i.e., do not require reconstitution, andare suitable for conventional sterilization process. The presentinvention satisfies these needs.

SUMMARY OF THE INVENTION

The present invention relates to reduction of flake-like aggregation ininjectable nanoparticulate active agent compositions. The compositioncomprises a nanoparticulate active agent, at least one surfacestabilizer and a flake-like aggregation reducing agent. The surfacestabilizer can be adsorbed on or associated with the surface of theactive agent particles. The nanoparticulate active agent particles havean effective average particle size of less than about 2000 nm.

A preferred dosage form of the invention is an injectablenanoparticulate active agent colloidal dispersion. The compositionoptionally comprises one or more pharmaceutically acceptable carriers,as well as any desired excipients. Preferably, the nanoparticulateactive agent colloidal dispersion is sterilized by passing through afilter having a pore size of 0.2 μm.

In one embodiment, the flake-like aggregation reducing agent is a sugaror polyol, such as sucrose, mannitol, or dextrose. In anotherembodiment, the flake-like aggregation reducing agent is a buffer, suchas a potassium phosphate buffer, a sodium phosphate buffer, or a sodiumacetate buffer. In one embodiment, the buffer results in the compositionhaving a pH above 7.0. Compositions according to the invention cancomprise more than one flake-like aggregation reducing agent, such as acombination of a sugar and a buffer.

In a related aspect, this invention further discloses a method of makingthe inventive nanoparticulate active agent compositions. Such a methodcomprises contacting the active agent with at least one surfacestabilizer in the presence of a flake-like aggregation reducing agentfor a time and under conditions sufficient to provide a nanoparticulateactive agent composition having an effective average particle size ofless than about 2000 nm. Alternatively, the surface stabilizer and/orflake-like aggregation reducing agent can be contacted with the activeagent particles either before, during, or after particle size reductionof the active agent particles.

In another aspect, the invention relates to a method for reducing theflake-like aggregates in a nanoparticulate composition. The methodcomprises preparing a nanoparticulate dispersion of an active agent andadding a flake-like aggregation reducing agent to the dispersion.Alternatively, a nanoparticulate dispersion of an active agent isprepared in the presence of a flake-like aggregation reducing agent.

In yet another aspect, the invention relates to a method for reducingthe flake-like aggregates in a nanoparticulate composition to meet therequirements of USP <788>. The method comprises preparing ananoparticulate dispersion of an active agent and adding a flake-likeaggregation reducing agent to the dispersion. Alternatively, ananoparticulate dispersion of an active agent is prepared in thepresence of a flake-like aggregation reducing agent.

In a further aspect, the invention relates to a method of treatmentusing a nanoparticulate active agent composition according to theinvention. The method comprises administering a nanoparticulate activeagent composition according to the invention, comprising at least onenanoparticulate active agent, at least one surface stabilizer, and atleast one flake-like aggregation reducing agent to a subject in need.The condition to be treated can be any condition susceptible totreatment by the active agent present in the nanoparticulatecomposition.

Both the foregoing summary of the invention and the following briefdescription of the drawings and detailed description of the inventionare exemplary and explanatory and are intended to provide furtherdetails of the invention as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the images of flake-like particulates in NanoCrystalColloidal Dispersion® NCD™ formulations.

FIG. 2 shows the particle size of a formulation containing 2.5%meloxicam and 0.5% PVP K17 at 25° C. and 40° C. storage for up to amonth.

FIG. 3 shows the particle size of a formulation containing 2.5%meloxicam, 0.5% PVP K17 and 0.25% NaDOC at 5° C., 25° C. and 40° C.storage for up to two months.

FIG. 4 shows the particle size of a formulation containing 2.5%meloxicam, 0.5% PVP K12 and 0.25% NaDOC at 5° C., 25° C. and 40° C.storage for up to two months.

FIG. 5 shows the particle size growth in the presence of 0.5% PVP atvariable concentration of NaDOC for over 3 months.

FIG. 6 shows the particle size growth in the presence of 0.75% PVP atvariable concentration of NaDOC for over 3 months.

FIG. 7 shows a magnified image of the NanoCrystal Colloidal Dispersion®NCD™ comprising 5% mannitol and 15% sucrose after it was filteredthrough an 8 μm filter at 200× magnification.

FIGS. 8A-8B shows the images of an NanoCrystal Colloidal Dispersion®NCD™ formulation without sugar (A) and an NanoCrystal ColloidalDispersion® NCD™ formulation comprising sugar (B). The particulateaggregates are observed in (A) but not in (B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to reduction of flake-like aggregation innanoparticulate compositions. The composition comprises ananoparticulate active agent having an effective average particle sizeof less than about 2000 nm, at least one surface stabilizer adsorbed onor associated with the surface of the active agent particles and aflake-like aggregation reducing agent.

As taught in the '684 patent, not every combination of surfacestabilizer and active agent will result in a stable nanoparticulatecomposition. The particulate matter test performed on somenanocrystalline formulations using the filtration/microscopy method asper USP<788> found high level of “flake-like particulates” as shown inFIG. 1. These formulations fail to meet the USP<788> criteria.

One problem present in prior art formulations of injectable dosage formsis the need for solubilizing solvents, such as cremofor, or in aqueoussolvents that may contain co-solvents and/or involve harsh pHconditions, all which may present significant toxicity uponadministration. Such formulations may still require large injectionvolumes, which further adds to the toxicity of the drug product.Injectable dosage forms of poorly soluble drugs that can be administeredin aqueous based media in high concentration without requiring harsh pHconditions or co-solvents are highly desirable.

An exemplary active agent that can be utilized in the formulations ofthe invention is meloxicam. Meloxicam (CAS No. 71125-38-7), chemicallyknown as4-hydroxy-2-methyl-N-(5-methyl-2-thiazolyl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide,has an empiric formula of C₁₄H₁₃N₃O₄S₂ and a molecular weight of351.403. Meloxicam is a pale yellow crystalline powder with no odor.Practically insoluble in water. The melting point is 254° C.

Meloxicam belongs to the family of nonsteroidal anti-inflammatory drugs(NSAIDs) and is used to relieve the symptoms of arthritis, primarydysmenorrheal, fever, and as an analgesic, especially where there is aninflammatory component.

Development of a nanoparticulate colloidal dispersion of meloxicamsuitable for injection encountered difficulty because it contained“flake-like” particulates ranging from 10-600 μm, which failed to meetthe USP<788> standard.

Thus, the discovery of the present invention is surprising in that aflake-like aggregation reducing agent could be successfully used toreduce aggregation of the particles of the nanoparticulate active agent.The obtained nanoparticulate active agent composition meets the criteriaof USP<788> and is suitable for injection.

I. Definitions

The present invention is described herein using several definitions, asset forth below and throughout the application.

As used herein, “about” will be understood by persons of ordinary skillin the art and will vary to some extent on the context in which it isused. If there are uses of the term which are not clear to persons ofordinary skill in the art given the context in which it is used, “about”will mean up to plus or minus 10% of the particular term.

“Nanoparticulate active agents” as defined herein have an effectiveaverage particle size of less than about 2 microns.

“Pharmaceutically acceptable” as used herein refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

As used herein with reference to stable drug particles, “stable”includes, but is not limited to, one or more of the followingparameters: (1) that the active agent particles do not appreciablyflocculate or agglomerate due to interparticle attractive forces, orotherwise significantly increase in particle size over time; (2) thatthe physical structure of the active agent particles is not altered overtime, such as by conversion from an amorphous phase to crystalline phaseor convert from one polymorph and/or hydrate to another; (3) that theactive agent particles are chemically stable; and/or (4) where theactive agent has not been subject to a heating step at or above themelting point of the active agent in the preparation of thenanoparticles of the invention.

“Therapeutically effective amount” as used herein with respect to anactive agent dosage, shall mean that dosage that provides the specificpharmacological response for which the active agent is administered in asignificant number of subjects in need of such treatment. It isemphasized that “therapeutically effective amount,” administered to aparticular subject in a particular instance will not always be effectivein treating the diseases described herein, even though such dosage isdeemed a ‘therapeutically effective amount’ by those skilled in the art.It is to be further understood that active agent dosages are, inparticular instances, measured as oral dosages, or with reference toactive agent levels as measured in blood.

II. Compositions

The compositions of the invention comprise a nanoparticulate activeagent, at least one surface stabilizer adsorbed to or associated withthe surface of the active agent, and at least one flake-like aggregationreducing agent. In addition, the compositions can comprise one or moresecondary flake-like aggregation reducing agent. Surface stabilizersuseful herein physically adhere to or associate with the surface of thenanoparticulate active agent but do not chemically react with the activeagent or itself. Individual molecules of the surface stabilizer areessentially free of intermolecular cross-linkages.

The present invention also includes nanoparticulate active agentcompositions formulated into compositions together with one or morenon-toxic physiologically acceptable carriers, adjuvants, or vehicles,collectively referred to as carriers.

A. Active Agents

The nanoparticles of the invention comprise at least one active,therapeutic, or diagnostic agent, collectively referred to as a “drug.”A therapeutic agent can be a pharmaceutical agent, including biologicssuch as proteins, peptides, and nucleotides, or a diagnostic agent, suchas a contrast agent, including x-ray contrast agents.

The active agent exists as a crystalline phase, an amorphous phase, asemi-amorphous phase, a semi-crystalline phase, or mixtures thereof. Thecrystalline phase differs from a non-crystalline or amorphous phasewhich results from precipitation techniques, such as those described inEP Patent No. 275,796.

The invention can be practiced with a wide variety of active agents. Theactive agent is preferably present in an essentially pure form, ispoorly soluble, and is dispersible in at least one liquid dispersionmedia. By “poorly soluble” it is meant that the active agent has asolubility in a liquid dispersion media of less than about 30 mg/mL,less than about 20 mg/mL, less than about 10 mg/mL, or less than about 1mg/mL. Useful liquid dispersion medias include, but are not limited to,water, aqueous salt solutions, safflower oil, and solvents such asethanol, t-butanol, hexane, and glycol. A preferred liquid dispersionmedia is water.

Two or more active agents can be used in combination.

1. Active Agents Generally

The active agent can be selected from a variety of known classes ofdrugs, including, for example, nutraceuticals, COX-2 inhibitors,retinoids, anticancer agents, NSAIDS, proteins, peptides, nucleotides,anti-obesity drugs, nutraceuticals, dietary supplements, carotenoids,corticosteroids, elastase inhibitors, anti-fungals, oncology therapies,anti-emetics, analgesics, cardiovascular agents, anti-inflammatoryagents, anthelmintics, anti-arrhythmic agents, antibiotics (includingpenicillins), anticoagulants, antidepressants, antidiabetic agents,antiepileptics, antihistamines, antihypertensive agents, antimuscarinicagents, antimycobacterial agents, antineoplastic agents,immunosuppressants, antithyroid agents, antiviral agents, anxiolytics,sedatives (hypnotics and neuroleptics), astringents, beta-adrenoceptorblocking agents, blood products and substitutes, cardiac inotropicagents, contrast media, corticosteroids, cough suppressants(expectorants and mucolytics), diagnostic agents, diagnostic imagingagents, diuretics, dopaminergics (antiparkinsonian agents),haemostatics, immunological agents, lipid regulating agents, musclerelaxants, parasympathomimetics, parathyroid calcitonin andbiphosphonates, prostaglandins, radio-pharmaceuticals, sex hormones(including steroids), anti-allergic agents, stimulants and anoretics,sympathomimetics, thyroid agents, vasodilators, and xanthines.

Examples of representative active agents useful in this inventioninclude, but are not limited to, acyclovir, alprazolam, altretamine,amiloride, amiodarone, benztropine mesylate, bupropion, cabergoline,candesartan, cerivastatin, chlorpromazine, ciprofloxacin, cisapride,clarithromycin, clonidine, clopidogrel, cyclobenzaprine, cyproheptadine,delavirdine, desmopressin, diltiazem, dipyridamole, dolasetron,enalapril maleate, enalaprilat, famotidine, felodipine, furazolidone,glipizide, irbesartan, ketoconazole, lansoprazole, loratadine, loxapine,mebendazole, mercaptopurine, milrinone lactate, minocycline,mitoxantrone, nelfinavir mesylate, nimodipine, norfloxacin, olanzapine,omeprazole, penciclovir, pimozide, tacrolimus, quazepam, raloxifene,rifabutin, rifampin, risperidone, rizatriptan, saquinavir, sertraline,sildenafil, acetyl-sulfisoxazole, temazepam, thiabendazole, thioguanine,trandolapril, triamterene, trimetrexate, troglitazone, trovafloxacin,verapamil, vinblastine sulfate, mycophenolate, atovaquone, proguanil,ceftazidime, cefuroxime, etoposide, terbinafine, thalidomide,fluconazole, amsacrine, dacarbazine, teniposide, and acetylsalicylate.In one embodiment, the active agent is meloxicam.

Exemplary nutraceuticals and dietary supplements are disclosed, forexample, in Roberts et al., Nutraceuticals: The Complete Encyclopedia ofSupplements, Herbs, Vitamins, and Healing Foods (American NutraceuticalAssociation, 2001), which is specifically incorporated by reference. Anutraceutical or dietary supplement, also known as a phytochemical orfunctional food, is generally any one of a class of dietary supplements,vitamins, minerals, herbs, or healing foods that have medical orpharmaceutical effects on the body. Exemplary nutraceuticals or dietarysupplements include, but are not limited to, lutein, folic acid, fattyacids (e.g., DHA and ARA), fruit and vegetable extracts, vitamin andmineral supplements, phosphatidylserine, lipoic acid, melatonin,glucosamine/chondroitin, Aloe Vera, Guggul, glutamine, amino acids(e.g., iso-leucine, leucine, lysine, methionine, phenylanine, threonine,tryptophan, and valine), green tea, lycopene, whole foods, foodadditives, herbs, phytonutrients, antioxidants, flavonoid constituentsof fruits, evening primrose oil, flax seeds, fish and marine animaloils, and probiotics. Nutraceuticals and dietary supplements alsoinclude bio-engineered foods genetically engineered to have a desiredproperty, also known as “pharmafoods.”

2. Anticancer Active Agents

Useful anticancer agents are preferably selected from alkylating agents,antimetabolites, natural products, hormones and antagonists, andmiscellaneous agents, such as radiosensitizers.

Examples of alkylating agents include: (1) alkylating agents having thebis-(2-chloroethyl)-amine group such as, for example, chlormethine,chlorambucile, melphalan, uramustine, mannomustine,extramustinephoshate, mechlore-thaminoxide, cyclophosphamide,ifosfamide, and trifosfamide; (2) alkylating agents having a substitutedaziridine group such as, for example, tretamine, thiotepa, triaziquone,and mitomycine; (3) alkylating agents of the alkyl sulfonate type, suchas, for example, busulfan, piposulfan, and piposulfam; (4) alkylatingN-alkyl-N-nitrosourea derivatives, such as, for example, carmustine,lomustine, semustine, or streptozotocine; and (5) alkylating agents ofthe mitobronitole, dacarbazine and procarbazine type.

Examples of antimetabolites include: (1) folic acid analogs, such as,for example, methotrexate; (2) pyrimidine analogs such as, for example,fluorouracil, floxuridine, tegafur, cytarabine, idoxuridine, andflucytosine; and (3) purine derivatives such as, for example,mercaptopurine, thioguanine, azathioprine, tiamiprine, vidarabine,pentostatin, and puromycine.

Examples of natural products include: (1) vinca alkaloids, such as, forexample, vinblastine and vincristine; (2) epipodophylotoxins, such as,for example, etoposide and teniposide; (3) antibiotics, such as, forexample, adriamycine, daunomycine, doctinomycin, daunorubicin,doxorubicin, mithramycin, bleomycin, and mitomycin; (4) enzymes, suchas, for example, L-asparaginase; (5) biological response modifiers, suchas, for example, alpha-interferon; (6) camptothecin; (7) taxol; and (8)retinoids, such as retinoic acid.

Examples of hormones and antagonists include: (1) adrenocorticosteroids,such as, for example, prednisone; (2) progestins, such as, for example,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate; (3) estrogens, such as, for example, diethylstilbestrol andethinyl estradiol; (4) antiestrogens, such as, for example, tamoxifen;(5) androgens, such as, for example, testosterone propionate andfluoxymesterone; (6) antiandrogens, such as, for example, flutamide; and(7) gonadotropin-releasing hormone analogs, such as, for example,leuprolide.

Examples of miscellaneous agents include: (1) radiosensitizers, such as,for example, 1,2,4-benzotriazin-3-amine 1,4-dioxide (SR 4889) and1,2,4-benzotriazine-7-amine 1,4-dioxide (WIN 59075); (2) platinumcoordination complexes such as cisplatin and carboplatin; (3)anthracenediones, such as, for example, mitoxantrone; (4) substitutedureas, such as, for example, hydroxyurea; and (5) adrenocorticalsuppressants, such as, for example, mitotane and aminoglutethimide.

In addition, the anticancer agent can be an immunosuppressive drug, suchas, for example, cyclosporine, azathioprine, sulfasalazine, methoxsalen,and thalidomide.

The anticancer agent can also be a COX-2 inhibitor.

3. Analgesic Active Agents

An analgesic can be, for example, an NSAID or a COX-2 inhibitor.

Exemplary NSAIDS that can be formulated in compositions of the inventioninclude, but are not limited to, suitable nonacidic and acidiccompounds. Suitable nonacidic compounds include, for example,nabumetone, tiaramide, proquazone, bufexamac, flumizole, epirazole,tinoridine, timegadine, and dapsone. Suitable acidic compounds include,for example, carboxylic acids and enolic acids. Suitable carboxylic acidNSAIDs include, for example: (1) salicylic acids and esters thereof,such as aspirin, diflunisal, benorylate, and fosfosal; (2) acetic acids,such as phenylacetic acids, including diclofenac, alclofenac, andfenclofenac; (3) carbo- and heterocyclic acetic acids such as etodolac,indomethacin, sulindac, tolmetin, fentiazac, and tilomisole; (4)propionic acids, such as carprofen, fenbufen, flurbiprofen, ketoprofen,oxaprozin, suprofen, tiaprofenic acid, ibuprofen, naproxen, fenoprofen,indoprofen, and pirprofen; and (5) fenamic acids, such as flufenamic,mefenamic, meclofenamic, and niflumic. Suitable enolic acid NSAIDsinclude, for example: (1) pyrazolones such as oxyphenbutazone,phenylbutazone, apazone, and feprazone; and (2) oxicams such aspiroxicam, sudoxicam, isoxicam, and tenoxicam.

Exemplary COX-2 inhibitors that can be formulated in combination withthe nanoparticulate nimesulide composition of the invention include, butare not limited to, celecoxib (SC-58635, CELEBREX®, Pharmacia/Searle &Co.), rofecoxib (MK-966, L-748731, VIOXX®, Merck & Co.), meloxicam(MOBIC®, co-marketed by Abbott Laboratories, Chicago, Ill., andBoehringer Ingelheim Pharmaceuticals), valdecoxib (BEXTRA®, G.D. Searle& Co.), parecoxib (G.D. Searle & Co.), etoricoxib (MK-663; Merck),SC-236 (chemical name of4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl)]benzenesulfonamide;G.D. Searle & Co., Skokie, Ill.); NS-398(N-(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide; TaishoPharmaceutical Co., Ltd., Japan); SC-58125 (methyl sulfonespiro(2.4)hept-5-ene I; Pharmacia/Searle & Co.); SC-57666(Pharmacia/Searle & Co.); SC-558 (Pharmacia/Searle & Co.); SC-560(Pharmacia/Searle & Co.); etodolac (Lodine®, Wyeth-Ayerst Laboratories,Inc.); DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulfonyl)phenyl2(5H)-furanone); monteleukast (MK-476), L-745337((5-methanesulphonamide-6-(2,4-difluorothio-phenyl)-1-indanone),L-761066, L-761000, L-748780 (all Merck & Co.); DUP-697(5-Bromo-2-(4-fluorophenyl)-3-(4-(methylsulfonyl)phenyl; DuPont MerckPharmaceutical Co.); PGV 20229(1-(7-tert.-butyl-2,3-dihydro-3,3-dimethylbenzo(b)furan-5-yl)-4-cyclopropylbutan-1-one;Procter & Gamble Pharmaceuticals); iguratimod (T-614;3-formylamino-7-methylsulfonylamino-6-phenoxy-4H-1-benzopyran-4-one;Toyama Corp., Japan); BF 389 (Biofor, USA); CL 1004 (PD 136095), PD136005, PD 142893, PD 138387, and PD 145065 (allParke-Davis/Warner-Lambert Co.); flurbiprofen (ANSAID®; Pharmacia &Upjohn); nabumetone (FELAFEN®, SmithKline Beecham, plc); flosulide (CGP28238; Novartis/Ciba Geigy); piroxicam (FELDANE®, Pfizer); diclofenac(VOLTAREN® and CATAFLAM®, Novartis); lumiracoxib (COX-189; Novartis); D1367 (Celltech Chiroscience, plc); R 807 (3 benzoyldifluoromethaneulfonanilide, diflumidone); JTE-522 (Japan Tobacco, Japan); FK-3311(4′-Acetyl-2′-(2,4-difluorophenoxy)methanesulfonanilide), FK 867, FR140423, and FR 115068 (all Fujisawa, Japan); GR 253035 (Glaxo Wellcome);RWJ 63556 (Johnson & Johnson); RWJ 20485 (Johnson & Johnson); ZK 38997(Schering); S 2474((E)-(5)-(3,5-di-tert-butyl-4-hydroxybenzylidene)-2-ethyl-1,2-isothiazolidine-1,1-dioxideindomethacin; Shionogi & Co., Ltd., Japan); zomepirac analogs, such asRS 57067 and RS 104897 (Hoffmann La Roche); RS 104894 (Hoffmann LaRoche); SC 41930 (Monsanto); pranlukast (SB 205312, Ono-1078, ONON®,ULTAIR®; SmithKline Beecham); SB 209670 (SmithKline Beecham); and APHS(heptinylsulfide).

B. Surface Stabilizers

The compositions of the invention include one or more surfacestabilizers. The surface stabilizers of the invention are preferablyadsorbed on, or associated with, the surface of the active agentparticles. The surface stabilizers especially useful herein preferablydo not chemically react with the active agent particles or itself.Preferably, individual molecules of the auxiliary surface stabilizer areessentially free of intermolecular cross-linkages.

Two or more surface stabilizers can be employed in the compositions andmethods of the invention.

Suitable surface stabilizers can preferably be selected from knownorganic and inorganic pharmaceutical excipients. Such excipients includevarious polymers, low molecular weight oligomers, natural products, andsurfactants. Preferred surface stabilizers include nonionic, anionic,cationic and zwitterionic compounds or surfactants.

Examples of useful nonionic stabilizers, including but not limited to,dextran, gum acacia, tragacanth, glycerol monostearate, cetostearylalcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylenealkyl ethers (e.g., macrogol ethers such as cetomacrogol 1000),polyoxyentylene alkyl esters (e.g. Myrj®), polyoxyethylene castor oilderivatives, polyoxyethylene sorbitan fatty acid esters (e.g., thecommercially available Tweens® such as e.g., Tween 20® and Tween 80®(ICI Speciality Chemicals)); polyethylene glycols (e.g., Carbowaxs 3550®and 934® (Union Carbide)), polyoxyethylene stearates, methylcellulose,hydroxyethylcellulose, hydroxypropyl celluloses (e.g., HPC, HPC-SL, andHPC-L), hydroxypropyl methylcellulose (HPMC),hydroxypropylmethyl-cellulose phthalate, noncrystalline cellulose,triethanolamine, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol, superione, and triton),poloxamers (e.g., Pluronics F68® and F108®, which are block copolymersof ethylene oxide and propylene oxide); poloxamines (e.g., Tetronic908®, also known as Poloxamine 908®, which is a tetrafunctional blockcopolymer derived from sequential addition of propylene oxide andethylene oxide to ethylenediamine (BASF Wyandotte Corporation,Parsippany, N.J.)); Tetronic 1508® (T-1508) (BASF WyandotteCorporation), alkyl phenol ethylene oxide (Triton X-100, Triton X-405,etc., Dow Chemical), Crodestas F-110®, which is a mixture of sucrosestearate and sucrose distearate (Croda Inc.);p-isononylphenoxypoly-(glycidol), also known as Olin-1OG® or Surfactant10-G® (Olin Chemicals, Stamford, Conn.); Crodestas SL-40® (Croda, Inc.);and SA9OHCO, which is C₁₈H₃₇CH₂C(O)N(CH₃)—CH₂(CHOH)₄(CH₂OH)₂ (EastmanKodak Co.); decanoyl-N-methylglucamide; n-decyl β-D-glucopyranoside;n-decyl β-D-maltopyranoside; n-dodecyl β-D-glucopyranoside; n-dodecylβ-D-maltoside; heptanoyl-N-methylglucamide;n-heptyl-β-D-glucopyranoside; n-heptyl β-D-thioglucoside; n-hexylβ-D-glucopyranoside; nonanoyl-N-methylglucamide; n-noylβ-D-glucopyranoside; octanoyl-N-methylglucamide;n-octyl-β-D-glucopyranoside; octyl β-D-thioglucopyranoside;PEG-derivatized phospholipid, PEG-derivatized cholesterol, PEG-derivatized cholesterol derivative, PEG-derivatized vitamin A,PEG-derivatized vitamin E, PEG-derivatized castor oil (Cremafor), randomcopolymers of vinyl pyrrolidone and vinyl acetate (an example would bePlasdone S-630, ISP).

Preferred nonionic stabilizers include, but are not limited to,Hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose gradeHPC-SL, Polyvinylpyrrolidones, Kollidone K12 (BASF) or Plasdone® C-12(ISP Technologies, Inc.) Kollidone K17 (BASF)—Plasdone® C-17 (ISPTechnologies, Inc). Kollidone K29/32 (BASF)—Plasdone® C-29/32 (ISPTechnologies, Inc. (USA), block copolymers based on ethylene oxide andpropylene oxide—Poloxamers sold under the tradename Pluronics® by BASF(sold as Lutrols® in EU), specifically Pluronic® F 68 a.k.a. poloxamer188, Pluronic® F 108 a.k.a. poloxamer 338, Pluronic® F 127 a.k.apoloxamer. 407, copolymer of vinylpyrrolidone and vinylacetate—Copovidone sold under the tradename Plasdone® S-630 (ISPTechnologies, Inc), distearyl palmitate glyceryl, polyoxyethylenesorbitan fatty acid esters such as polyoxyethylene 20 sorbitanmonolaurate, polysorbate 20 a.k.a. Tween® 20 by ICI Americas,polyoxyethylene 20 sorbitan monopalmitate, polysorbate 40″ a.k.a. Tween®40 by ICI Americas, polyoxyethylene 20 sorbitan monooleate, polysortbate80 a.k.a. Tween® 80 by ICI Americas, macrogol 15hydroxystearate—Solutol® 15 BASF, Tyloxapol and Cremaphor.

Examples of useful anionic stabilizers, including but not limited to,fatty acids as well as their salt forms such as oleic acid, stearicacid, palmitic acid, lauric acid, myristic acid, calcium stearate. Othercommon anionic stabilizers include sodium dodecylsulfate, Duponol P®,which is a sodium lauryl sulfate (DuPont), carboxymethylcellulosecalcium, carboxymethylcellulose sodium, dialkylesters of sodiumsulfosuccinic acid (e.g., Aerosol OT®, which is a dioctyl ester ofsodium sulfosuccinic acid (DOSS) (American Cyanamid)), Triton X-200®,which is an alkyl aryl polyether sulfonate (Union Carbide). Salt formsof bile acids are also useful as anionic stabilizers, such as sodiumdeoxycholate, sodium cholate, sodium chenodeoxycholate, sodiumdehydrocholate, disuccinylursodeoxycholic acid bisodic salt, sodiumhyodeoxycholate, sodium ursodeoxycholate.

Preferred anionic stabilizers include, but are not limited to, dioctylsodium succinate (DOSS) sodium lauryl sulfate (SLS) a.k.a. sodiumdodecyl sulfate (SDS) and sodium deoxycholate.

Examples of useful cationic surface stabilizers include but are notlimited to polymers, biopolymers, poly-n-methylpyridinium, anthryulpyridinium chloride, cationic phospholipids, chitosan, polylysine,polyvinylimidazole, polybrene, polymethylmethacrylatetrimethylammoniumbromide bromide (PMMTMABr), hexyldesyltrimethylammoniumbromide (HDMAB), and polyvinylpyrrolidone-2-dimethylaminoethylmethacrylate dimethyl sulfate.

Other useful cationic stabilizers include, but are not limited to,cationic lipids, sulfonium, phosphonium, and quarternary ammoniumcompounds, such as stearyltrimethylammonium chloride,benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride or bromide, coconut methyl dihydroxyethyl ammoniumchloride or bromide, dodecyl trimethyl ammonium bromide, decyl triethylammonium chloride, decyl dimethyl hydroxyethyl ammonium chloride orbromide, C₁₂₋₁₅dimethyl hydroxyethyl ammonium chloride or bromide,coconut dimethyl hydroxyethyl ammonium chloride or bromide, myristyltrimethyl ammonium methyl sulphate, lauryl dimethyl benzyl ammoniumchloride or bromide, lauryl dimethyl (ethenoxy)₄ ammonium chloride orbromide, N-alkyl (C₁₂₋₁₈)dimethylbenzyl ammonium chloride, N-alkyl(C₁₄₋₁₈)dimethyl-benzyl ammonium chloride, N-tetradecylidmethylbenzylammonium chloride monohydrate, dimethyl didecyl ammonium chloride,N-alkyl and (C₁₂₋₁₄) dimethyl 1-napthylmethyl ammonium chloride,trimethylammonium halide, alkyl-trimethylammonium salts anddialkyl-dimethylammonium salts, lauryl trimethyl ammonium chloride,ethoxylated alkyamidoalkyldialkylammonium salt and/or an ethoxylatedtrialkyl ammonium salt, dialkylbenzene dialkylammonium chloride,N-didecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl(C₁₂-₁₄) dimethyl1-naphthylmethyl ammonium chloride and dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C₁₂, C₁₅, C₁₇ trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, poly-diallyldimethylammoniumchloride (DADMAC), dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride (ALIQUAT 336™), POLYQUAT 10™,tetrabutylammonium bromide, benzyl trimethylammonium bromide, cholineesters (such as choline esters of fatty acids), benzalkonium chloride,stearalkonium chloride compounds (such as stearyltrimonium chloride andDi-stearyldimonium chloride), cetyl pyridinium bromide or chloride,halide salts of quaternized polyoxyethylalkylamines, MIRAPOL™ andALKAQUAT™ (Alkaril Chemical Company), alkyl pyridinium salts; amines,such as alkylamines, dialkylamines, alkanolamines,polyethylenepolyamines, N,N-dialkylaminoalkyl acrylates, and vinylpyridine, amine salts, such as lauryl amine acetate, stearyl amineacetate, alkylpyridinium salt, and alkylimidazolium salt, and amineoxides; imide azolinium salts; protonated quaternary acrylamides;methylated quaternary polymers, such as poly[diallyl dimethylammoniumchloride] and poly-[N-methyl vinyl pyridinium chloride]; and cationicguar.

Such exemplary cationic surface stabilizers and other useful cationicsurface stabilizers are described in J. Cross and E. Singer, CationicSurfactants: Analytical and Biological Evaluation (Marcel Dekker, 1994);P. and D. Rubingh (Editor), Cationic Surfactants: Physical Chemistry(Marcel Dekker, 1991); and J. Richmond, Cationic Surfactants: OrganicChemistry, (Marcel Dekker, 1990).

Particularly preferred nonpolymeric primary stabilizers are anynonpolymeric compound, such benzalkonium chloride, a carbonium compound,a phosphonium compound, an oxonium compound, a halonium compound, acationic organometallic compound, a quarternary phosphorous compound, apyridinium compound, an anilinium compound, an immonium compound, ahydroxylammonium compound, a primary ammonium compound, a secondaryammonium compound, a tertiary ammonium compound, and quarternaryammonium compounds of the formula NR₁R₂R₃R₄ ⁽⁺⁾. For compounds of theformula NR₁R₂R₃R₄ ⁽⁺⁾:

-   -   (i) none of R₁-R₄ are CH₃;    -   (ii) one of R₁-R₄ is CH₃;    -   (iii) three of R₁-R₄ are CH₃;    -   (iv) all of R₁-R₄ are CH₃;    -   (v) two of R₁-R₄ are CH₃, one of R₁-R₄ is C₆H₅CH₂, and one of        R₁-R₄ is an alkyl chain of seven carbon atoms or less;    -   (vi) two of R₁-R₄ are CH₃, one of R₁-R₄ is C₆H₅CH₂, and one of        R₁-R₄ is an alkyl chain of nineteen carbon atoms or more;    -   (vii) two of R₁-R₄ are CH₃ and one of R₁-R₄ is the group        C₆H₅(CH₂)_(n), where n>1;    -   (viii) two of R₁-R₄ are CH₃, one of R₁-R₄ is C₆H₅CH₂, and one of        R₁-R₄ comprises at least one heteroatom;    -   (ix) two of R₁-R₄ are CH₃, one of R₁-R₄ is C₆H₅CH₂, and one of        R₁-R₄ comprises at least one halogen;    -   (x) two of R₁-R₄ are CH₃, one of R₁-R₄ is C₆H₅CH₂, and one of        R₁-R₄ comprises at least one cyclic fragment;    -   (xi) two of R₁-R₄ are CH₃ and one of R₁-R₄ is a phenyl ring; or    -   (xii) two of R₁-R₄ are CH₃ and two of R₁-R₄ are purely aliphatic        fragments.

Such compounds include, but are not limited to, behenalkonium chloride,benzethonium chloride, cetylpyridinium chloride, behentrimoniumchloride, lauralkonium chloride, cetalkonium chloride, cetrimoniumbromide, cetrimonium chloride, cethylamine hydrofluoride,chlorallylmethenamine chloride (Quaternium-15), distearyldimoniumchloride (Quaternium-5), dodecyl dimethyl ethylbenzyl ammoniumchloride(Quaternium-14), Quaternium-22, Quaternium-26, Quaternium-18hectorite, dimethylaminoethylchloride hydrochloride, cysteinehydrochloride, diethanolammonium POE (10) oletyl ether phosphate,diethanolammonium POE (3)oleyl ether phosphate, tallow alkoniumchloride, dimethyl dioctadecylammoniumbentonite, stearalkonium chloride,domiphen bromide, denatonium benzoate, myristalkonium chloride,laurtrimonium chloride, ethylenediamine dihydrochloride, guanidinehydrochloride, pyridoxine HCl, iofetamine hydrochloride, megluminehydrochloride, methylbenzethonium chloride, myrtrimonium bromide,oleyltrimonium chloride, polyquaternium-1, procainehydrochloride,cocobetaine, stearalkonium bentonite, stearalkoniumhectonite, stearyltrihydroxyethyl propylenediamine dihydrofluoride, tallowtrimoniumchloride, and hexadecyltrimethyl ammonium bromide.

Examples of zwitterionic stabilizers include but are not limited toproteins, phospholipids, zwitterionic polymers and zwitterionicsurfactant molecules. Examples of proteins albumin, including but notlimited to human serum albumin and bovine serum albumin, gelatin,casein, lysozyme. Exmaples of phospholipids include phosphotidylcholine,lecithin. The proteins and peptides are zwitteronic may morph intocationic or anionic depending on the pH of the medium they are exposedto. In this embodiment, it should be understood that in the consideredpH range, these are molecules are zwitterionic.

Most of these surface stabilizers are known pharmaceutical excipientsand are described in detail in the Handbook of PharmaceuticalExcipients, published jointly by the American Pharmaceutical Associationand The Pharmaceutical Society of Great Britain (The PharmaceuticalPress, 5^(th) ed., 2005), specifically incorporated by reference. Thesurface stabilizers are commercially available and/or can be prepared bytechniques known in the art.

C. Flake-Like Aggregation Reducing Agent

The present invention is directed to the surprising discovery thatreduction of flake-like aggregation can be achieved by adding a sugar ina nanoparticulate active agent composition or by increasing the pH levelof a nanoparticulate active agent to basic conditions.

According to the present invention, the buffered formulation is at a pHthat is suitably high enough to reduce flake-like aggregation. Thecompositions of the invention have a pH level of about pH 7.0, about7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7,about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0,about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about9.7, about 9.8, about 9.9, about 10.0, about 10.1, about 10.2, about10.3, about 10.4, about 10.5, about 10.6, about 10.7, about 10.8, about10.9, about 11.0, about 11.1, about 11.2, about 11.3, about 11.4, about11.5, about 11.6, about 11.7, about 11.8, about 11.9, about 12.0, about12.1, about 12.2, about 12.3, about 12.4, about 12.5, about 12.6, about12.7, about 12.8, about 12.9, about 13.0, about 13.1, about 13.2, about13.3, about 13.4, about 13.5, about 13.6, about 13.7, about 13.8, about13.9, or about 14.0, or above about 14.0. In one embodiment, the pH ofthe formulation of the invention is in a range selected from the groupconsisting of pH about 9.0-about 10.0, about 10.0-about 11.0, about11.0-about 12.0, about 12.0-about 14.0. In another embodiment, the pH ofthe formulation is in the range of pH about 9.5-about 11.0. In yetanother embodiment, the pH of the formulation is in the range of pHabout 7.0-about 9.5.

In the context of this invention, a flake-like aggregation reducingagent is defined as an agent that is capable of reducing the flake-likeaggregation in a nanoparticulate active agent composition. Exemplaryflake-like aggregation reducing agents include sugars, sugar alcoholsand buffers. Exemplary sugars and sugar alcohols includes, but are notlimited to, sucrose, fructose, glucose, erythritol, isomalt, mannitol,sorbitol, xylitol, sorbitol, and dextrose. Any pH buffering systemsuitable for I.V. or I.M. administration may be used, such as but notlimited to a phosphate buffer, an acetate buffer or a citrate buffer. Insome embodiments, the buffer is a potassium phosphate buffer, a sodiumphosphate buffer, or a sodium acetate buffer.

D. Other Pharmaceutical Excipients

Pharmaceutical compositions according to the invention may also compriseone or more binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art.

Examples of filling agents include lactose monohydrate, lactoseanhydrous, and various starches; examples of binding agents includevarious celluloses and cross-linked polyvinylpyrrolidone,microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102,microcrystalline cellulose, and silicified microcrystalline cellulose(ProSolv SMCC™).

Suitable lubricants, including agents that act on the flowability of thepowder to be compressed, may include colloidal silicon dioxide, such asAerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate,and silica gel.

Examples of sweeteners may include any natural or artificial sweetener,such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, andacesulfame. Examples of flavoring agents are Magnasweet® (trademark ofMAFCO), bubble gum flavor, and fruit flavors, and the like.

Examples of preservatives include potassium sorbate, methylparaben,propylparaben, benzoic acid and its salts, other esters ofparahydroxybenzoic acid such as butylparaben, alcohols such as ethyl orbenzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride.

Suitable diluents include pharmaceutically acceptable inert fillers,such as microcrystalline cellulose, lactose, dibasic calcium phosphate,saccharides, and/or mixtures of any of the foregoing. Examples ofdiluents include microcrystalline cellulose, such as Avicel® PH101 andAvicel® PH102; lactose such as lactose monohydrate, lactose anhydrous,and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®;mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof.

Examples of effervescent agents include effervescent couples such as anorganic acid and a carbonate or bicarbonate. Suitable organic acidsinclude, for example, citric, tartaric, malic, fumaric, adipic,succinic, and alginic acids and anhydrides and acid salts. Suitablecarbonates and bicarbonates include, for example, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, andarginine carbonate. Alternatively, only the sodium bicarbonate componentof the effervescent couple may be present.

E. Nanoparticulate Active Agent Particle Size

The compositions of the invention contain nanoparticulate active agentparticles which have an effective average particle size of less thanabout 2000 nm (i.e., 2 microns). In other embodiments of the invention,the active agent particles have a size of less than about 1900 nm, lessthan about 1800 nm, less than about 1700 nm, less than about 1600 nm,less than about 1500 nm, less than about 1400 nm, less than about 1300nm, less than about 1200 nm, less than about 1100 nm, less than about1000 nm, less than about 990 nm, less than about 980 nm, less than about970 nm, less than about 960 nm, less than about 950 nm, less than about940 nm, less than about 930 nm, less than about 920 nm, less than about910 nm, less than about 900 nm, less than about 890 nm, less than about880 nm, less than about 870 nm, less than about 860 nm, less than about850 nm, less than about 840 nm, less than about 830 nm, less than about820 nm, less than about 810 nm, less than about 800 nm, less than about790 nm, less than about 780 nm, less than about 770 nm, less than about760 nm, less than about 750 nm, less than about 740 nm, less than about730 nm, less than about 720 nm, less than about 710 nm, less than about700 nm, less than about 690 nm, less than about 680 nm, less than about670 nm, less than about 660 nm, less than about 650 nm, less than about640 nm, less than about 630 nm, less than about 620 nm, less than about610 nm, less than about 600 nm, less than about 590 nm, less than about580 nm, less than about 570 nm, less than about 560 nm, less than about550 nm, less than about 540 nm, less than about 530 nm, less than about520 nm, less than about 510 nm, less than about 500 nm, less than about490 nm, less than about 480 nm, less than about 470 nm, less than about460 nm, less than about 450 nm, less than about 440 nm, less than about430 nm, less than about 420 nm, less than about 410 nm, less than about400 nm, less than about 390 nm, less than about 380 nm, less than about370 nm, less than about 360 nm, less than about 350 nm, less than about340 nm, less than about 330 nm, less than about 320 nm, less than about310 nm, less than about 300 nm, less than about 290 nm, less than about280 nm, less than about 270 nm, less than about 260 nm, less than about250 nm, less than about 240 nm, less than about 230 nm, less than about220 nm, less than about 210 nm, less than about 200 nm, less than about190 nm, less than about 180 nm, less than about 170 nm, less than about160 nm, less than about 150 nm, less than about 140 nm, less than about130 nm, less than about 120 nm, less than about 110 nm, less than about100, less than about 75 nm, or less than about 50 nm, as measured bylight-scattering methods, microscopy, or other appropriate methods.

By “an effective average particle size of less than about 2000 nm” it ismeant that at least 50% of the active agent particles have a particlesize less than the effective average, by weight (or by other suitablemeasurement techniques, such as by volume, number, etc.), i.e., lessthan about 2000 nm, 1900 nm, 1800 nm, etc., when measured by theabove-noted techniques. In other embodiments of the invention, at leastabout 60%, at least about 70%, at least about 80% at least about 90%, atleast about 95%, or at least about 99% of the active agent particleshave a particle size less than the effective average, i.e., less thanabout 2000 nm, 1900 nm, 1800 nm, etc.

In the present invention, the value for D50 of a nanoparticulate activeagent composition is the particle size below which 50% of the activeagent particles fall, by weight. Similarly, D90 and D99 are the particlesizes below which 90% and 99%, respectively, of the active agentparticles fall, by weight (or by other suitable measurement techniques,such as by volume, number, etc.).

F. Nanoparticulate Active Agent Particulate Matter

One or more representative samplings of a particulate composition can beassayed according to the USP methods by using commercially-availableparticle sizing and counting machines. These commercially availableparticle counting machines are designed to ensure sample particulateinjectable solutions comply with the USP <788> particle sizingrequirements.

1. USP <788> Particulate Matter in Injections

The following text is taken from USP<788> guidelines for preparingparticulate drug products for injections and Chapter 788 in its entiretyis incorporated herein by reference.

This general chapter is harmonized with the corresponding texts of theEuropean Pharmacopoeia and/or the Japanese Pharmacopoeia. Thesepharmacopeias have undertaken not to make any unilateral change to thisharmonized chapter.

Portions of the present general chapter text that are national USP text,and therefore not part of the harmonized text, are marked with symbols(*_(*)) to specify this fact.

Particulate matter in injections and parenteral infusions consists ofmobile undissolved particles, other than gas bubbles, unintentionallypresent in the solutions.

For the determination of particulate matter, two procedures, Method 1(Light Obscuration Particle Count Test) and Method 2 (MicroscopicParticle Count Test), are specified hereinafter. When examininginjections and parenteral infusions for sub-visible particles Method 1is preferably applied. However, it may be necessary to test somepreparations by the light obscuration particle count test followed bythe microscopic particle count test to reach a conclusion on conformanceto the requirements.

Not all parenteral preparations can be examined for sub-visibleparticles by one or both of these methods. When Method 1 is notapplicable, e.g. in case of preparations having reduced clarity orincreased viscosity, the test should be carried out according to Method2. Emulsions, colloids, and liposomal preparations are examples.Similarly, products that produce air or gas bubbles when drawn into thesensor may also require microscopic particle count testing. If theviscosity of the preparation to be tested is sufficiently high so as topreclude its examination by either test method, a quantitative dilutionwith an appropriate diluent may be made to decrease viscosity, asnecessary, to allow the analysis to be performed.

The results obtained in examining a discrete unit or group of units forparticulate matter cannot be extrapolated with certainty to other unitsthat remain untested. Thus, statistically sound sampling plans must bedeveloped if valid inferences are to be drawn from observed data tocharacterize the level of particulate matter in a large group of units.

Method 1. Light Obscuration Particle Count Test

Use a suitable apparatus based on the principle of light blockage whichallows an automatic determination of the size of particles and thenumber of particles according to size. The definition for particle-freewater is provided in Reagent Specifications under Reagents, Indicatorsand Solution section.

The apparatus is calibrated using dispersions of spherical particles ofknown sizes between 10 μm and 25 μm. These standard particles aredispersed in particle-free water.

Care must be taken to avoid aggregation of particles during dispersion.System suitability can be verified by using the USP Particle Count RS(<11>).

General Precautions

The test is carried out under conditions limiting particulate matter,preferably in a laminar-flow cabinet.

Very carefully wash the glassware and filtration equipment used, exceptfor the membrane filters, with a warm detergent solution and rinse withabundant amounts of water to remove all traces of detergent. Immediatelybefore use, rinse the equipment from top to bottom, outside and theninside, with particle-free water.

Take care not to introduce air bubbles into the preparation to beexamined, especially when fractions of the preparation are beingtransferred to the container in which the determination is to be carriedout.

To check that the environment is suitable for the test, that theglassware is properly cleaned and that the water to be used isparticle-free, the following test is carried out: determine theparticulate matter in 5 samples of particle-free water, each of 5 ml,according to the method described below. If the number of particles of10 μm or greater size exceeds 25 for the combined 25 ml, the precautionstaken for the test are not sufficient. The preparatory steps must berepeated until the environment, glassware and water are suitable for thetest.

Method

Mix the contents of the sample by slowly inverting the container 20times successively. If necessary, cautiously remove the sealing closure.Clean the outer surfaces of the container opening using a jet ofparticle-free water and remove the closure, avoiding any contaminationof the contents. Eliminate gas bubbles by appropriate measures such asallowing to stand for 2 min or sonicating.

For large-volume parenterals, single units are tested. For small-volumeparenterals less than 25 ml in volume, the contents of 10 or more unitsis combined in a cleaned container to obtain a volume of not less than25 ml; the test solution may be prepared by mixing the contents of asuitable number of vials and diluting to 25 ml with particle-free wateror with an appropriate particle-free solvent when particle-free water isnot suitable. Small-volume parenterals having a volume of 25 ml or moremay be tested individually.

Powders for parenteral use are reconstituted with particle-free water orwith an appropriate particle-free solvent when particle-free water isnot suitable.

The number of test specimens must be adequate to provide a statisticallysound assessment. For large-volume parenterals or for small-volumeparenterals having a volume of 25 ml or more, fewer than 10 units may betested, based on an appropriate sampling plan.

Remove four portions, each of not less than 5 ml, and count the numberof particles equal to or greater than 10 μm and 25 μm. Disregard theresult obtained for the first portion, and calculate the mean number ofparticles for the preparation to be examined.

Evaluation

For preparations supplied in containers with a nominal volume of morethan 100 ml, apply the criteria of test 1.A.

For preparations supplied in containers with a nominal volume of lessthan 100 ml, apply the criteria of test 1.B.

For preparations supplied in containers with a nominal volume of 100 ml,apply the criteria of test 1.B [Note: Test 1.A is used in the JapanesePharmacopoeia]

If the average number of particles exceeds the limits, test thepreparation by the Microscopic Particle Count Test.

Test 1.A—Solutions for parenteral infusion or solutions for injectionsupplied in containers with a nominal content of more than 100 mL.

The preparation complies with the test if the average number ofparticles present in the units tested does not exceed 25 per mL equal toor greater than 10 μm and does not exceed 3 per mL equal to or greaterthan 25 μm.

Test 1.B—Solutions for parenteral infusion or solutions for injectionsupplied in containers with a nominal content of less than 100 ml.

The preparation complies with the test if the average number ofparticles present in the units tested does not exceed 6000 per containerequal to or greater than 10 μm and does not exceed 600 per containerequal to or greater than 25 μm.

Method 2. Microscopic Particle Count Test

Use a suitable binocular microscope, filter assembly for retainingparticulate matter and membrane filter for examination.

The microscope is equipped with an ocular micrometer calibrated with anobjective micrometer, a mechanical stage capable of holding andtraversing the entire filtration area of the membrane filter, twosuitable illuminators to provide episcopic illumination in addition tooblique illumination, and is adjusted to 100±10 magnifications.

The ocular micrometer is a circular diameter graticule (see FIG. 1) andconsists of a large circle divided by crosshairs into quadrants,transparent and black reference circles 10 μm and 25 μm in diameter at100 magnifications, and a linear scale graduated in 10 μm increments. Itis calibrated using a stage micrometer that is certified by either adomestic or international standard institution. A relative error of thelinear scale of the graticule within ±2 per cent is acceptable. Thelarge circle is designated the graticule field of view (GFOV).

Two illuminators are required. One is an episcopic brightfieldilluminator internal to the microscope, the other is an external,focusable auxiliary illuminator adjustable to give reflected obliqueillumination at an angle of 10° to 20°.

The filter assembly for retaining particulate matter consists of afilter holder made of glass or other suitable material, and is equippedwith a vacuum source and a suitable membrane filter.

The membrane filter is of suitable size, black or dark gray in color,non-gridded or gridded, and 1.0 μm or finer in nominal pore size.

General Precautions

The test is carried out under conditions limiting particulate matter,preferably in a laminar-flow cabinet.

Very carefully wash the glassware and filter assembly used, except forthe membrane filter, with a warm detergent solution and rinse withabundant amounts of water to remove all traces of detergent. Immediatelybefore use, rinse both sides of the membrane filter and the equipmentfrom top to bottom, outside and then inside, with particle-free water.

In order to check that the environment is suitable for the test, thatthe glassware and the membrane filter are properly cleaned and that thewater to be used is particle-free, the following test is carried out:determine the particulate matter of a 50 ml volume of particle-freewater according to the method described below. If more than 20 particles10 μm or larger in size or if more than 5 particles 25 μm or larger insize are present within the filtration area, the precautions taken forthe test are not sufficient. The preparatory steps must be repeateduntil the environment, glassware, membrane filter and water are suitablefor the test.

2. USP <788> as Applied to the Invention

The present invention is not limited to the specific instruments orstrategies outlined in USP<788> for obtaining an approved particulatedrug product. For instance, the skilled person knows that variousaspects of the USP<788> “Microscopy” method can vary, such as the sizeof the pores of a filter, the source and direction of lightillumination, the type of color filter used to help visualize theparticles with the microscope, and the various different ways one canreduce the viscosity of a solution by dilution. For instance, theskilled person knows that the filter may have a filter pore size ofabout 5 μm in size, about 4 μm in size, about 3 μm in size, about 2 μmin size, or about 1 μm in size. In one embodiment, the filter pore sizeis about from 1-5 μm in size. Similarly, the skilled person knows thatthe microscopy platform onto which the sample is viewed may beilluminated from the top or bottom of the microscopy apparatus. In oneembodiment, the source of light illumination is from the top. Likewise,the skilled person knows that it is possible to change the color filterused to visualize the sample depending on the source and direction ofthe illumination. Thus, the skilled person knows of the availability ofdark, black, grey, or white filters for this purpose. In one embodiment,the filter is white. Thus, in one embodiment, the present inventioncontemplates obtaining a particulate drug product that complies with theUSP<788> size threshold requirements by using a filter that has a 5 μmpore size, illuminated from the top using a white color filter. Inanother embodiment, the skilled person knows how to dilute a solution toreduce viscosity in the USP<788> methods.

The skilled person would be able to determine the optimal or suitableconditions for retaining the nanoparticulate active agent in anon-aggregated form, so that the formulation complies with USP<788>.Thus, nanoparticulate active agent compositions of the present inventiondescribed herein contains (i) no more than 6,000 particles that aregreater than 10 μm in size and (ii) no more than 600 particles that aregreater than 25 μm in size, under the Light Obscuration test ofUSP<788>. And in another embodiment, the nanoparticulate active agentcompositions of the present invention contains no more than (i) 3,000particles that are greater than 10 μm in size and (ii) no more than 300particles that are greater than 25 μm in size, under use of theMicroscopy method of USP<788>.

The present invention is not limited to the particular size thresholdsset forth in USP<788>. Thus in one embodiment, an injectable formulationof the present invention has fewer than about 1000 particles larger than25 μm, fewer than about 900 particles larger than 25 μm, fewer thanabout 800 particles larger than 25 μm, fewer than about 700 particleslarger than 25 μm, fewer than about 600 particles larger than 25 μm,fewer than about 500 particles larger than 25 μm, fewer than about 400particles larger than 25 μm, fewer than about 300 particles larger than25 μm, fewer than about 250 particles larger than 25 μm, fewer thanabout 200 particles larger than 25 μm, fewer than about 150 particleslarger than 25 μm, fewer than about 100 particles larger than 25 μm, orfewer than about 50 particles larger than 25 μm.

In another embodiment, an injectable formulation of the presentinvention has fewer than about 10000 particles larger than 10 μm, fewerthan about 9000 particles larger than 10 μm, fewer than about 8000particles larger than 10 μm, fewer than about 7000 particles larger than10 μm, fewer than about 6000 particles larger than 10 μm, fewer thanabout 5000 particles larger than 10 μm, fewer than about 4000 particleslarger than 10 μm, fewer than about 3000 particles larger than 10 μm,fewer than about 2000 particles larger than 10 μm, fewer than about 1000particles larger than 10 μm, fewer than about 900 particles larger than10 μm, fewer than about 800 particles larger than 10 μm, fewer thanabout 700 particles larger than 10 μm, fewer than about 600 particleslarger than 10 μm, fewer than about 500 particles larger than 10 μm,fewer than about 400 particles larger than 10 μm, fewer than about 300particles larger than 10 μm, fewer than about 200 particles larger than10 μm, fewer than about 175 particles larger than 10 μm, fewer thanabout 150 particles larger than 10 μm, fewer than about 100 particleslarger than 10 μm, fewer than about 75 particles larger than 10 μm,fewer than about 50 particles larger than 10 μm, fewer than about 25particles larger than 10 μm, fewer than about 15 particles larger than10 μm, fewer than about 10 particles larger than 10 μm, fewer than about5 particles larger than 10 μm, fewer than about 3 particles larger than10 μm, or essentially no particles larger than 10 μm.

G. Concentration of Nanoparticulate Active Agent, Surface Stabilizer,and Flake-Like Aggregation Reducing Agent

The relative amounts of active agent, surface stabilizer and flake-likeaggregation reducing agent can vary widely. The optimal amount of theindividual components can depend, for example, upon the particularactive agent and surface stabilizer selected, the hydrophilic lipophilicbalance (HLB), melting point, and the surface tension of water solutionsof the surface stabilizer, etc.

The concentration of the surface stabilizer can vary from about 0.5% toabout 99.999%, from about 5.0% to about 99.9%, or from about 10% toabout 99.5%, by weight, based on the total combined dry weight of the atleast one active agent and at least one surface stabilizer, notincluding other excipients.

The concentration of the at least one active agent can vary from about99.5% to about 0.001%, from about 95% to about 0.1%, or from about 90%to about 0.5%, by weight, based on the total combined dry weight of theactive agent and at least one surface stabilizer, not including otherexcipients.

III. Methods of Making Nanoparticulate Active Agent Formulations

The nanoparticulate active agent compositions of the invention,comprising at least one surface stabilizer and at least one flake-likeaggregation reducing agent, can be made using, for example, milling orattrition (including but not limited to wet milling), homogenization,precipitation, freezing, template emulsion techniques, supercriticalfluid techniques, nano-electrospray techniques, or any combinationthereof. Exemplary methods of making nanoparticulate active agentcompositions are described in the '684 patent. Methods of makingnanoparticulate active agent compositions are also described in U.S.Pat. No. 5,518,187 for “Method of Grinding Pharmaceutical Substances;”U.S. Pat. No. 5,718,388 for “Continuous Method of GrindingPharmaceutical Substances;” U.S. Pat. No. 5,862,999 for “Method ofGrinding Pharmaceutical Substances;” U.S. Pat. No. 5,665,331 for“Co-Microprecipitation of Nanoparticulate Pharmaceutical Agents withCrystal Growth Modifiers;” U.S. Pat. No. 5,662,883 for“Co-Microprecipitation of Nanoparticulate Pharmaceutical Agents withCrystal Growth Modifiers;” U.S. Pat. No. 5,560,932 for“Microprecipitation of Nanoparticulate Pharmaceutical Agents;” U.S. Pat.No. 5,543,133 for “Process of Preparing X-Ray Contrast CompositionsContaining Nanoparticles;” U.S. Pat. No. 5,534,270 for “Method ofPreparing Stable Drug Nanoparticles;” U.S. Pat. No. 5,510,118 for“Process of Preparing Therapeutic Compositions ContainingNanoparticles;” and U.S. Pat. No. 5,470,583 for “Method of PreparingNanoparticle Compositions Containing Charged Phospholipids to ReduceAggregation,” all of which are specifically incorporated by reference.

A. Milling to obtain Nanoparticulate Active Agent Dispersions

Milling the active agent to obtain a nanoparticulate colloidaldispersion comprises dispersing active agent particles in a liquiddispersion media in which the active agent is poorly soluble, followedby applying mechanical means in the presence of grinding media to reducethe particle size of the active agent to the desired effective averageparticle size. The dispersion media can be, for example, water,safflower oil, ethanol, t-butanol, glycerin, polyethylene glycol (PEG),hexane, or glycol. Water is a preferred dispersion media.

The active agent particles are preferably reduced in size in thepresence of at least one surface stabilizer. Alternatively, the activeagent particles can be contacted with at least one surface stabilizereither during or after attrition. One or more flake-like aggregationreducing agent may be added before, during, or after attrition. Othercompounds, such as a diluent, can be added to the composition before,during, or after the size reduction process. Dispersions can bemanufactured continuously or in a batch mode.

The grinding media can comprise particles that are preferablysubstantially spherical in shape, e.g., beads, consisting essentially ofpolymeric or copolymeric resin. Alternatively, the grinding media cancomprise a core having a coating of a polymeric or copolymeric resinadhered thereon.

In general, suitable polymeric or copolymeric resins are chemically andphysically inert, substantially free of metals, solvent, and monomers,and of sufficient hardness and friability to enable them to avoid beingchipped or crushed during grinding. Suitable polymeric or copolymericresins include crosslinked polystyrenes, such as polystyrene crosslinkedwith divinylbenzene; styrene copolymers; polycarbonates; polyacetals,such as Delrin™ (E.I. du Pont de Nemours and Co.); vinyl chloridepolymers and copolymers; polyurethanes; polyamides;poly(tetrafluoroethylenes), e.g., Teflon® (E.I. du Pont de Nemours andCo.), and other fluoropolymers; high density polyethylenes;polypropylenes; cellulose ethers and esters such as cellulose acetate;polyhydroxymethacrylate; polyhydroxyethyl acrylate; andsilicone-containing polymers such as polysiloxanes and the like. Thepolymer can be biodegradable. Exemplary biodegradable polymers orcopolymers include poly(lactides), poly(glycolide) copolymers oflactides and glycolide, polyanhydrides, poly(hydroxyethyl methacrylate),poly(imino carbonates), poly(N-acylhydroxyproline)esters,poly(N-palmitoyl hydroxyproline) esters, ethylene-vinyl acetatecopolymers, poly(orthoesters), poly(caprolactones), andpoly(phosphazenes). For biodegradable polymers or copolymers,contamination from the media itself advantageously can metabolize invivo into biologically acceptable products that can be eliminated fromthe body.

The grinding media preferably ranges in size from about 0.01 to about 3mm. For fine grinding, the grinding media is preferably from about 0.02to about 2 mm, and more preferably from about 0.03 to about 1 mm insize.

The polymeric or copolymeric resin can have a density from about 0.8 toabout 3.0 g/cm³.

In a preferred grinding process the active agent particles are madecontinuously. Such a method comprises continuously introducing an activeagent composition according to the invention into a milling chamber,contacting the active agent composition according to the invention withgrinding media while in the chamber to reduce the active agent particlesize of the composition according to the invention, and continuouslyremoving the nanoparticulate active agent composition from the millingchamber.

The grinding media is separated from the milled nanoparticulate activeagent composition according to the invention using conventionalseparation techniques, in a secondary process such as by simplefiltration, sieving through a mesh filter or screen, and the like. Otherseparation techniques such as centrifugation may also be employed.

B. Precipitation to Obtain Nanoparticulate Active Agent Compositions

Another method of forming the desired nanoparticulate active agentcomposition is by microprecipitation. This is a method of preparingstable nanoparticulate active agent dispersions of poorly soluble activeagents in the presence of one or more surface stabilizers and one ormore colloid stability enhancing surface active agents free of any tracetoxic solvents or solubilized heavy metal impurities. Such a methodcomprises, for example: (1) dissolving the poorly soluble active agentin a suitable solvent; (2) adding the formulation from step (1) to asolution comprising at least one surface stabilizer and optionally oneor more flake-like aggregation reducing agent, to form a clear solution;and (3) precipitating the formulation from step (2) using an appropriatenon-solvent. The method can be followed by removal of any formed salt,if present, by dialysis or diafiltration and concentration of thedispersion by conventional means.

C. Homogenization to Obtain Nanoparticulate Active Agent Compositions

Exemplary homogenization methods of preparing active agentnanoparticulate active agent compositions are described in U.S. Pat. No.5,510,118, for “Process of Preparing Therapeutic Compositions ContainingNanoparticles.”

Such a method comprises dispersing active agent particles in a liquiddispersion media in which the active agent is poorly soluble, followedby subjecting the dispersion to homogenization to reduce the particlesize of the active agent to the desired effective average particle size.The active agent particles can be reduced in size in the presence of atleast one surface stabilizer and, optionally, one or more flake-likeaggregation reducing agent. Alternatively, the active agent particlescan be contacted with at least one surface stabilizer and one or moreflake-like aggregation reducing agent either during or after attrition.Other compounds, such as a diluent, can be added to the compositioneither before, during, or after the size reduction process. Dispersionscan be manufactured continuously or in a batch mode.

D. Cryogenic Methodologies to Obtain Nanoparticulate Active AgentCompositions

Another method of forming the desired nanoparticulate active agentcomposition is by spray freezing into liquid (SFL). This technologycomprises an organic or organoaqueous solution of an active agent withone or more surface stabilizers. One or more flake-like aggregationreducing agent can be added either before, during, or after particlesize reduction. The composition is injected into a cryogenic liquid,such as liquid nitrogen. The droplets of active agent solution freeze ata rate sufficient to minimize crystallization and particle growth, thusformulating nanostructured active agent particles. Depending on thechoice of solvent system and processing conditions, the nanoparticulateactive agent particles can have varying particle morphology. In theisolation step, the nitrogen and solvent are removed under conditionsthat avoid agglomeration or ripening of the active agent particles.

As a complementary technology to SFL, ultra rapid freezing (URF) mayalso be used to created equivalent nanostructured active agent particleswith greatly enhanced surface area. URF comprises an organic ororganoaqueous solution of active agent with stabilizers onto a cryogenicsubstrate.

E. Emulsion Methodologies to Obtain Nanoparticulate Active AgentCompositions

Another method of forming the desired nanoparticulate active agentcomposition is by template emulsion. Template emulsion createsnanostructured active agent particles with controlled particle sizedistribution and rapid dissolution performance. The method comprises anoil-in-water emulsion that is prepared, then swelled with a non-aqueoussolution comprising active agent and one or more surface stabilizers.One or more flake-like aggregation reducing agent can be added eitherbefore, during, or after particle size reduction. The particle sizedistribution of the active agent is a direct result of the size of theemulsion droplets prior to loading with the active agent, a propertywhich can be controlled and optimized in this process. Furthermore,through selected use of solvents and stabilizers, emulsion stability isachieved with no or suppressed Ostwald ripening. Subsequently, thesolvent and water are removed, and the stabilized nanostructured activeagent particles are recovered. Various active agent particlemorphologies can be achieved by appropriate control of processingconditions.

F. Supercritical Fluid Methods of Making Active Agent Nanoparticles

Nanoparticulate active agent compositions can also be made in methodsutilizing supercritical fluids. In such a method, the active agent isdissolved in a solution or vehicle which can also contain at least onesurface stabilizer. One or more flake-like aggregation reducing agentcan be added either before, during, or after particle size reduction.The solution and a supercritical fluid are then co-introduced into aparticle formation vessel. If a surface stabilizer was not previouslyadded to the vehicle, it can be added to the particle formation vesselThe temperature and pressure are controlled, such that dispersion andextraction of the vehicle occur substantially simultaneously by theaction of the supercritical fluid. Chemicals described as being usefulas supercritical fluids include carbon dioxide, nitrous oxide, sulphurhexafluoride, xenon, ethylene, chlorotrifluoromethane, ethane, andtrifluoromethane.

Examples of known supercritical methods of making nanoparticles includeInternational Patent Application No. WO 97/14407 to Pace et al.,published on Apr. 24, 1997, which refers to particles of water insolublebiologically active compounds with an average size of 100 nm to 300 nmprepared by dissolving the compound in a solution and then spraying thesolution into compressed gas, liquid, or supercritical fluid in thepresence of appropriate surface stabilizers.

Similarly, U.S. Pat. No. 6,406,718 to Cooper et al. describes a methodfor forming a particulate fluticasone propionate product comprising theco-introduction of a supercritical fluid and a vehicle containing atleast fluticasone propionate in solution or suspension into a particleformation vessel, the temperature and pressure in which are controlled,such that dispersion and extraction of the vehicle occur substantiallysimultaneously by the action of the supercritical fluid. Chemicalsdescribed as being useful as supercritical fluids include carbondioxide, nitrous oxide, sulphur hexafluoride, xenon, ethylene,chlorotrifluoromethane, ethane, and trifluoromethane. The supercriticalfluid may optionally contain one or more modifiers, such as methanol,ethanol, ethyl acetate, acetone, acetonitrile or any mixture thereof. Asupercritical fluid modifier (or co-solvent) is a chemical which, whenadded to a supercritical fluid, changes the intrinsic properties of thesupercritical fluid in or around the critical point. According to Cooperet al., the fluticasone propionate particles produced usingsupercritical fluids have a particle size range of 1 to 10 microns,preferably 1 to 5 microns.

G. Nano-Electrospray Techniques Used to Obtain Nanoparticulate ActiveAgent Compositions

In electrospray ionization a liquid is pushed through a very smallcharged, usually metal, capillary. This liquid contains the desiredactive agent dissolved in a large amount of solvent, which is usuallymuch more volatile than the active agent (e.g., analyte). Volatileacids, bases or buffers are often added to this solution as well. Theanalyte exists as an ion in solution either in a protonated form or asan anion. As like charges repel, the liquid pushes itself out of thecapillary and forms a mist or an aerosol of small droplets about 10 μmacross. This jet of aerosol droplets is at least partially produced by aprocess involving the formation of a Taylor cone and a jet from the tipof this cone. A neutral carrier gas, such as nitrogen gas, is sometimesused to help nebulize the liquid and to help evaporate the neutralsolvent in the small droplets. As the small droplets evaporate,suspended in the air, the charged analyte molecules are forced closertogether. The drops become unstable as the similarly charged moleculescome closer together and the droplets once again break up. This isreferred to as Coulombic fission because it is the repulsive Coulombicforces between charged analyte molecules that drive it. This processrepeats itself until the analyte is free of solvent and is a lone ion.

In nanotechnology the electrospray method may be employed to depositsingle active agent particles on surfaces. This is accomplished byspraying colloids and ensuring that on average there is not more thanone particle per droplet. Consequent drying of the surrounding solventresults in an aerosol stream of single active agent particles. Here theionizing property of the process is not crucial for the application butmay be put to use in electrostatic precipitation of the particles.

IV. Methods of Using Nanoparticulate Active Agent Formulations

The nanoparticulate active agent compositions of the present inventioncan be administered to humans and animals via any conventional meansincluding, but not limited to, orally, rectally, ocularly, parenterally(intravenous, intramuscular, or subcutaneous), intracisternally,pulmonary, intravaginally, intraperitoneally, locally (powders,ointments or drops), or as a buccal or nasal spray.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, and the like), suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions,and by the use of surfactants.

The nanoparticulate active agent compositions may also contain adjuvantssuch as preserving, wetting, emulsifying, and dispensing agents.Prevention of the growth of microorganisms can be ensured by variousantibacterial and antifungal agents, such as parabens, chlorobutanol,phenol, sorbic acid, and the like. It may also be desirable to includeisotonic agents, such as sugars, sodium chloride, and the like.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, such as aluminummonostearate and gelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activeagent is admixed with at least one of the following: (a) one or moreinert excipients (or carrier), such as sodium citrate or dicalciumphosphate; (b) fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and silicic acid; (c) binders, such ascarboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose and acacia; (d) humectants, such as glycerol; (e) disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain complex silicates, and sodium carbonate; (f)solution retarders, such as paraffin; (g) absorption accelerators, suchas quaternary ammonium compounds; (h) wetting agents, such as cetylalcohol and glycerol monostearate; (i) adsorbents, such as kaolin andbentonite; and (j) lubricants, such as talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. For capsules, tablets, and pills, the dosage forms may alsocomprise buffering agents.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active agent, the liquid dosage forms may comprise inertdiluents commonly used in the art, such as water or other solvents,solubilizing agents, and emulsifiers. Exemplary emulsifiers are ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propyleneglycol, 1,3-butyleneglycol,dimethylformamide, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethyleneglycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Actual dosage levels of active agent in the nanoparticulate compositionsof the invention may be varied to obtain an amount of active agent thatis effective to obtain a desired therapeutic response for a particularcomposition and method of administration. The selected dosage leveltherefore depends upon the desired therapeutic effect, the route ofadministration, the potency of the administered active agent, thedesired duration of treatment, and other factors.

Dosage unit compositions may contain such amounts of such submultiplesthereof as may be used to make up the daily dose. It will be understood,however, that the specific dose level for any particular patient willdepend upon a variety of factors including the body weight, generalhealth, sex, diet, time and route of administration, potency of theadministered active agent, rates of absorption and excretion,combination with other active agents, and the severity of the particulardisease being treated.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including a U.S. patent, are specificallyincorporated by reference.

The formulations in the examples that follow were also investigatedusing a light microscope. Here, “stable” nanoparticulate dispersions(uniform Brownian motion) were readily distinguishable from “aggregated”dispersions (relatively large, nonuniform particles without motion).

EXAMPLE 1

The purpose of this example was to prepare a nanoparticulate meloxicamdispersion suitable for injection that met the USP <788> standard andtest the stability of the dispersion.

A slurry of 20% (w/w) meloxicam (supplied by anonima Materie SinteticheE Affini Spa) and 4% (w/w) polyvinyl pyrrolidone (Povidone Kollidon® 17PF) was milled for 4 hours in a NanoMill-2 system using PolyMill® 500 μmgrinding media. The meloxicam particle size stability under controlledconditions was monitored over time.

It was observed that under all stability conditions, the nanopartiuclatemeloxicam dispersion prepared as described above had levels offlake-like particulates that were too numerous to count and consequentlyfailed to meet USP<788> criteria. FIG. 1 shows the particulates found inthe NanoCrystal Colloidal Dispersion® NCD™.

EXAMPLE 2

The purpose of this example was to screen nanoparticulate meloxicamformulations for the stability of particle size and particulate matter.

The nanoparticulate meloxicam formulations as outlined in FIGS. 2-6 andfurther summarized in Table 1 below were made as described in Example 1.The resulting dispersions were tested at 5° C., 25° C. or 40° C. for upto 3 months.

TABLE 1 Storage time Condition Dmean D50 D90 Formulation Days ° C. nm nmnm 2.5% meloxicam, 0.5% Pvp K17 30 25 157 95 334 30 40 295 259 476 2.5%meloxicam, 0.5% Pvp K17, 0.25% NaDOC 60 5 99 89 121 60 25 101 90 127 6040 106 91 145 2.5% meloxicam, 0.25% Pvp K17, 0.25% Tween 80 29 5 273 256417 29 25 468 446 710 29 40 585 566 858 2.5% meloxicam, 0.5% Pvp K12,0.25% NaDOC 62 5 102 89 130 62 25 104 91 137 62 40 106 91 148 2.5%meloxicam, 0.5% Tween 20, 0.5% Span 20 29 5 113 89 225 29 25 451 425 69629 40 548 537 825 2.3% meloxicam, 0.5% Pvp K17, 2.3% PEG400, 0.23% 0 NA123 105 214 NaDOC 2.5% meloxicam, 0.5% Pvp K17, 0.25% NaDOC, 200 mM 7 5139 Sodium Phosphate pH 9 2.5% meloxicam, 0.5% Pvp K17, 0.125% NaDOC, 145 139 125 mM Sodium Phosphate pH 7.5 2.3% meloxicam, 0.46% Pvp K17,0.23% NaDOC, 50 mM 7 5 102 Sodium Phosphate pH 6 2.7% meloxicam, 0.54%Pvp K17, 200 mM Sodium 14 5 164 Phosphate pH 9 2.5% meloxicam, 0.5% PvpK17, 0.08% NaDOC, 0.1M 0 5 115 Potassium Phosphate pH 7.5 3.4%meloxicam, 0.68% Pvp K17, 0.23% NaDOC, 0.68% 0 5 220 Tween 80, 100 mMSodium Phosphate pH 8

Table 2 summarizes the aforementioned formulations and the levels ofparticulate matter. This table shows a need for further improvement insome of formulations to reduce the flake-like particulates.

TABLE 2 Storage Storage Particles Approx. Particles time ConditionObserved Retained Formulation Days ° C. (100 GFOV)* on 47 mm Filter 2.5%meloxicam, 0.5% Pvp K17, 0.125% NaDOC 7 5 575 9,976 7 25 127 2,203 7 40477 8,275 2.5% meloxicam, 0.38% Pvp K17, 0.19% NaDOC 7 5 1,096 19,015 725 5 87 7 40 224 3,886 2.5% meloxicam, 0.5% Pvp K17, 0.375% NaDOC 7 5118 2,047 7 25 78 1,353 7 40 TNTC N/A 2.5% meloxicam, 0.38% Pvp K17,0.38% NaDOC 7 5 70 1,214 7 25 45 781 7 40 182 3,158 2.5% meloxicam,0.75% Pvp K17, 0.125% NaDOC 0 NA 291 2019 2.5% meloxicam, 0.75% Pvp K17,0.25% NaDOC 7 5 242 4,198 7 25 8 139 7 40 72 1,249 2.5% meloxicam, 0.75%Pvp K17, 0.5% NaDOC 7 25 20 347 2.5% meloxicam, 0.5% Pvp K12, 0.125%NaDOC 7 5 168 2,915 7 25 292 5,066 7 40 199 3,452 2.5% meloxicam, 0.5%Pvp K12, 0.25% NaDOC 7 5 94 1,631 7 25 68 1,180 7 40 368 6,384 2.5%meloxicam, 0.5% Pvp K12, 0.375% NaDOC 7 5 58 1,006 7 25 98 1700 7 40 7862.5% meloxicam, 0.5% Pvp K17, 0.5% NaDOC 7 5 18 312 7 25 65 1,128 7 4071 1,232 2.5% meloxicam, 0.75% Pvp K17, 0.125% NaDOC 7 5 27 468 7 40 4718171 2.5% meloxicam, 0.75% Pvp K2, 0.25% NaDOC 7 5 23 399 7 25 385 6,6797 40 33 573 2.5% meloxicam, 0.75% Pvp K12, 0.375% NaDOC 7 25 89 1,5442.5% meloxicam, 0.75% Pvp K12, 0.5% NaDOC 0 NA 41 711 2.3% meloxicam,0.5% Pvp K17, 2.3% PEG400, 0.23% 0 NA could not NaDOC filter 2.5%meloxicam, 0.5% Pvp K17, 0.25% NaDOC, 5% 7 5 2 35 Sucrose 7 25 6 104 740 0 0 2.5% meloxicam, 0.5% Pvp K17, 0.25% NaDOC, 200 mM 7 5 107 SodiumPhosphate pH 9 2.5% meloxicam, 0.5% Pvp K17, 0.125% NaDOC, 14 5 7165 125mM Sodium Phosphate pH 7.5 2.3% meloxicam, 0.46% Pvp K17, 0.23% NaDOC,50 mM 7 5 103 Sodium Phosphate pH 6 2.7% meloxicam, 0.54% Pvp K17, 200mM Sodium 14 5 1145 Phosphate pH 9 2.5% meloxicam, 0.5% Pvp K17, 0.08%NaDOC, 0.1M 14 5 698 Potassium Phosphate pH 7.5 3.4% meloxicam, 0.68%Pvp K17, 0.23% NaDOC, 0.68% 7 5 1694 Tween 80, 100 mM Sodium PhosphatepH 8 Certain formulations in Table 2, such as the one formulationcontaining sucrose and ones that were pH adjusted with pH buffers showsignificant improvement in particulate matter levels

EXAMPLE 3

The purpose of this example was to test the stability of ananoparticulate meloxicam formulation comprising mannitol in terms ofmeloxicam particle size and particulate matter.

A slurry of 10% (w/w) meloxicam, 2.5% (w/w) PVP K17, 0.75% (w/w) NaDOCand 10% (w/w) mannitol in polymill 500 μm grinding media at 89% mediaload was milled for 4 hours using a DynoMill 300 mL chamber inrecirculation mode to obtain a nanopartiuclate dispersion of meloxicam.The nanoparticulate meloxicam dispersion was diluted to 5% meloxicam,1.25% PVP K17, 0.375% NaDOC, 5% mannitol and 15% sucrose with a 30%sucrose solution. The formulation was stored at 5° C. for 3 months andtested for particulate matter.

FIG. 7 shows a magnified image of the nanoparticulate meloxicamdispersion after it was filtered through an 8 μm filter at200×magnification. Only 6 flake-like particulates were counted on filterranging from 10-25 μm.

EXAMPLE 4

The purpose of this example was to screen nanoparticulate meloxicamformulations comprising sucrose and/or mannitol for the stability ofmeloxicam particle size and particulate matter.

Meloxicam formulations were milled in the presence of sucrose and/ormannitol on a DynoMill. After 7 to 90 days of storage at 5° C., 25° C.or 40° C., the formulations were tested for particle size andparticulate matter. The results are summarized in Tables 3 and 4,respectively.

TABLE 3 Storage Time Condition Dmean D50 D90 Formulation Days ° C. nm nmnm 2.5% meloxicam, 0.5% 90 5 109 93 189 PVP K-17, 0.25% NaDOC, 90 25 11496 199 5% Sucrose 90 40 118 101 204 2.5% meloxicam, 0.5% 90 5 156 141230 PVP K-17, 1.25% Sucrose, 90 25 155 142 227 1.25% Mannitol 90 40 162149 235 2.5% meloxicam, 0.5% 90 5 96 94 122 PVP K-17, 2.5% Mannitol, 9025 99 95 127 0.25% NaDOC 2.5% meloxicam, 0.5% 90 5 92 91 114 PVP K-17,1.25% Sucrose, 90 25 94 92 117 1.25% Mannitol, 0.25% 90 40 98 95 126NaDOC

TABLE 4 Storage Particles Approx. Particles Time Condition ObservedRetained Formulation Days ° C. (100 GFOV)* on 47 mm Filter 2.5%meloxicam, 0.5% 7 5 37 642 PVP K-17, 2.5% Mannitol, 7 25 52 902 0.25%NaDOC 7 40 204/165** 3539/2863** 2.5% meloxicam, 0.5% 7 25 75 1301  PVPK-17, 1.25% Sucrose, 1.25% 7 40 16 278 Mannitol, 0.25% NaDOC *GraticuleField of View. **Results obtained by two different analysts

It appeared that including sucrose and/or mannitol was able to reducethe flake-like aggregation in the formulations. Further screeningstudies were performed to determine the type and concentration of sugarin the formulations.

Additional meloxicam formulations were milled using either a DynoMillwith a 150 mL chamber or a NanoMill-01 with a 100 mL chamber. Theformulations comprising 2.5% meloxicam were stored at 5° C., 25° C. or40° C. for up to 3 months and counted for particulate matter. Theresults are summarized in Table 5.

TABLE 5 Storage NaDOC Sucrose Mannitol Temp. Particulate Counts SamplePVP (%) (%) (%) (%) (° C.) initial 1 wk 2 wks 1 mon 3 mons 1 0.5 K170.25 10 0 5 243 1006 1666 109 25 156 17 32 40 35 87 71 2 0.5 K17 0.25 50 5 295 746 191 57 25 69 69 121 40 69 555 116 3 0.5 K17 0.25 2.5 0 5 8502307 208 148 25 451 208 52 40 226 121 74 4 0.5 K17 0.25 1.25 0 5 1024 87330 89 25 69 17 35 40 52 347 69 5 0.5 K17 0.25 0 5 5 937 382 590 69 2587 139 102 40 52 295 992 6 0.5 K17 0.25 0 2.5 5 104 416 1284 63 25 330139 119 40 382 278 104 7 0.5 K17 0.25 0 1.25 5 243 1006 87 60 25 52 46829 40 1284 2082 81 8 0.75 K12  0.25 10 0 5 n/a 1 7 56 23 25 27 4 174 1340 13 9 34 10 9 0.5 K12 0.25 0 0 5 n/a 9368 25 5569 40 12665 10 0.5 K170.25 0 0 5 541 19015 25 87 40 3886 11 0.75 K12  0.25 0 0 5 3834 399 256679 40 573

EXAMPLE 5

The purpose of this example was to demonstrate the suitability ofnanoparticulate meloxicam formulations for a sterile filtration process,and thus suitable for parenteral use.

Formulations comprising 2.5% meloxicam were milled in the presence of0.5% PVP K17 or 0.75% PVP K12 and two different concentrations ofsucrose where successfully filtered through a sterilizing grade filter.The amount of filtered material is indicative of a pilot scale process.The test conditions and results are summarized in Table 6.

TABLE 6 API PVP K-12 PVP K-17 Sucrose NaDOC Mean PS Amount (%) (%) (%)(%) (%) (nm) filtered 2.50 — 0.50 10.0 0.25 72  1200 g 2.50 — 0.50 10.00.25 112 >5000 g 2.50 — 0.50 10.0 0.25 139 >5000 g 2.50 0.75 — 10.0 0.25139 >5000 g

The formulations comprising 0.5% PVP K17 showed no difference infilterability from the formulations comprising 0.75% PVP K12.

EXAMPLE 10

The purpose of this example was to evaluate the stability of meloxicamformulations with different pH levels in terms of meloxicam particlesize and particulate matter.

A slurry of 15% meloxicam and 3% PVP K17 was milled on a DynoMill at 89%media load at a speed of 4200 rpm for 60 minutes. 0%, 0.75% or 1.5% ofNaDOC was included in the milling slurry. Sodium phosphate buffers at aconcentration of 50 mM, 100 mM, 125 mM or 200 mM and at a pH level of pH6.0, 7.5 and 9.0 were used to adjust the pH of the formulations.Alternatively, potassium phosphate, such as potassium phosphate/NaOH atpH 7.5 was used. The formulations were generally adjusted with sodiumphosphate buffers unless otherwise noted.

The obtained nanoparticulate meloxicam dispersion was diluted to thefinal concentration of 2.5% meloxicam, 0.5% PVP K17 with sterile waterfor injection or with additional buffer. The stability results aresummarized in Table 7.

TABLE 7 Buffer % Particulate Particle Conc NaDOC NCD ™*** Dmean Countcount/ (mM) (Slurry) pH (nm) (unfiltered) 10 mL* 200 1.5 7.6 129 5/20 ml2 125 0.75 7.4 119 15/20 ml 8 50 1.5 6.5 102 168/20 ml 84  50 0 5.7 113TNTC/150 ml TNTC 125 0 7 123 46/225 ml 2 200 1.5 6.2 106 TNTC/20 ml TNTC** 0.5 7.2 115 137/20 ml 68  *Normalized to count/10 mL from previouscolumn ** pH was adjusted with potassium phosphate/NaOH ***NCD ™ =NanoCrystal Colloidal Dispersion ®

In general, samples with pH above 7 returned the lowest initialparticulate counts, from 2 to 68 per 10 ml sample; while the sampleshaving a pH of 6.5 or less had the highest particulate counts, from 84to too numerous to count per 10 mL of sample.

Two exemplary formulations with superior properties were identified:

-   (1) a formulation comprising 2.5% meloxicam, 0.5% PVP K17, and    0.125% NaDOC in buffer pH 7.5 at 125 mM, yielding a final    NanoCrystal Colloidal Dispersion® NCD™ pH of 7.4. The particulate    count was 15 flakes per 20 mL and-   (2) a formulation comprising 2.5% meloxicam, 0.5% PVP K17, and 0.25%    NaDOC in buffer pH 9 at 200 mM, yielding a final NanoCrystal    Colloidal Dispersion® NCD™ pH of 7.6. The particulate count was 5    per 20 mL of sample.

What is claimed is:
 1. An injectable, nanoparticulate meloxicamcomposition consisting of: (a) about 2.5% to about 5% (w/w) of meloxicamparticles having an effective average particle size of less than about400 nm; (b) about 0.5% to about 1.625% (w/w) of at least onenon-crosslinked surface stabilizer adsorbed on the surface of themeloxicam particles; (c) about 1.25% to about 20% (w/w) of a flake-likeaggregation reducing agent; wherein the flake-like aggregation reducingagent consists of sucrose; and (d) water; wherein the injectable,nanoparticulate meloxicam composition comprises no more than 3,000meloxicam particles that are greater than 10 μm in size and no more than300 active meloxicam particles that are greater than 25 μm in size whenmeasured in a nominal volume of 25 mL, and wherein meloxicam is the onlyactive agent in the composition, and wherein the composition is stableand suitable for injection after storage for at least 3 months at 25° C.2. The nanoparticulate meloxicam composition of claim 1, wherein themeloxicam particles have an effective average particle size of less thanabout 300 nm.
 3. The nanoparticulate meloxicam composition of claim 1,wherein the meloxicam particles have an effective average particle sizeof less than about 200 nm.
 4. The nanoparticulate meloxicam compositionof claim 1, wherein the surface stabilizer is selected from the groupconsisting of a non-ionic surface stabilizer, an ionic surfacestabilizer, an anionic surface stabilizer, a cationic surfacestabilizer, and a zwitterionic surface stabilizer.
 5. Thenanoparticulate meloxicam composition of claim 1, wherein the surfacestabilizer is selected from the group consisting of cetyl pyridiniumchloride, gelatin, casein, phosphatides, dextran, glycerol, gum acacia,cholesterol, tragacanth, stearic acid, benzalkonium chloride, calciumstearate, glycerol monostearate, cetostearyl alcohol, cetomacrogolemulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers,polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fattyacid esters, polyethylene glycols, dodecyl trimethyl ammonium bromide,polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodiumdodecyl sulfate, carboxymethylcellulose calcium, hydroxypropylcelluloses, hypromellose, carboxymethylcellulose sodium,methylcellulose, hydroxyethylcellulose, hypromellose phthalate,noncrystalline cellulose, magnesium aluminum silicate, triethanolamine,polyvinyl alcohol, polyvinylpyrrolidone,4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde, poloxamers; poloxamines, a charged phospholipid,dioctylsulfosuccinate, dialkylesters of sodium sulfosuccinic acid,sodium lauryl sulfate, alkyl aryl polyether sulfonates, mixtures ofsucrose stearate and sucrose distearate,p-isononylphenoxypoly-(glycidol), decanoyl-N-methylglucamide; n-decylβ-D-glucopyranoside; n-decyl β-D-maltopyranoside; n-dodecylβ-D-glucopyranoside; n-dodecyl β-D-maltoside;heptanoyl-N-methylglucamide; n-heptyl-β-D-glucopyranoside; n-heptylβ-D-thioglucoside; n-hexyl β-D-glucopyranoside;nonanoyl-N-methylglucamide; n-noyl β-D-glucopyranoside;octanoyl-N-methylglucamide; n-octyl-β-D-glucopyranoside; octylβ-D-thioglucopyranoside; lysozyme, PEG-phospholipid, PEG-cholesterol,PEG-cholesterol derivative, PEG-vitamin A, random copolymers of vinylacetate and vinyl pyrrolidone, a cationic polymer, a cationicbiopolymer, a cationic polysaccharide, a cationic cellulosic, a cationicalginate, a cationic nonpolymeric compound, a cationic phospholipids,cationic lipids, polymethylmethacrylate trimethylammonium bromide,sulfonium compounds, polyvinylpyrrolidone-2-dimethylaminoethylmethacrylate dimethyl sulfate, hexadecyltrimethyl ammonium bromide,phosphonium compounds, quarternary ammonium compounds,benzyl-di(2-chloroethyl)ethylammonium bromide, coconut trimethylammonium chloride, coconut trimethyl ammonium bromide, coconut methyldihydroxyethyl ammonium chloride, coconut methyl dihydroxyethyl ammoniumbromide, decyl triethyl ammonium chloride, decyl dimethyl hydroxyethylammonium chloride, decyl dimethyl hydroxyethyl ammonium chloridebromide, C12-15_dimethyl hydroxyethyl ammonium chloride, C12-15_dimethylhydroxyethyl ammonium chloride bromide, coconut dimethyl hydroxyethylammonium chloride, coconut dimethyl hydroxyethyl ammonium bromide,myristyl trimethyl ammonium methyl sulphate, lauryl dimethyl benzylammonium chloride, lauryl dimethyl benzyl ammonium bromide, lauryldimethyl (ethenoxy)4 ammonium chloride, lauryl dimethyl (ethenoxy)_4ammonium bromide, N-alkyl (C12-18)_dimethylbenzyl ammonium chloride,N-alkyl (C14-18)_dimethyl-benzyl ammonium chloride,N-tetradecylidmethylbenzyl ammonium chloride monohydrate, dimethyldidecyl ammonium chloride, N-alkyl and (C12-14) dimethyl 1-napthylmethylammonium chloride, trimethylammonium halide, alkyl-trimethylammoniumsalts, dialkyl-dimethylammonium salts, lauryl trimethyl ammoniumchloride, ethoxylated alkyamidoalkyldialkylammonium salt, an ethoxylatedtrialkyl ammonium salt, dialkylbenzene dialkylammonium chloride,N-didecyldimethyl ammonium chloride, N-tetradecyldimethylbenzylammonium, chloride monohydrate, N-alkyl_(C12-14) dimethyl1-naphthylmethyl ammonium chloride, dodecyldimethylbenzyl ammoniumchloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethylammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyldimethyl ammonium bromide, C12 trimethyl ammonium bromides, C15trimethyl ammonium bromides, C17 trimethyl ammonium bromides,dodecylbenzyl triethyl ammonium chloride, polydiallyldimethylammoniumchloride, dimethyl ammonium chlorides, alkyldimethylammoniumhalogenides, tricetyl methyl ammonium chloride, decyltrimethylammoniumbromide, dodecyltriethylammonium bromide, tetradecyltrimethylammoniumbromide, methyl trioctylammonium chloride, tetrabutylammonium bromide,benzyl trimethylammonium bromide, choline esters, benzalkonium chloride,stearalkonium chloride compounds, cetyl pyridinium bromide, cetylpyridinium chloride, halide salts of quaternizedpolyoxyethylalkylamines, alkyl pyridinium salts; amines, amine salts,amine oxides, imide azolinium salts, protonated quaternary acrylamides,methylated quaternary polymers, and cationic guar.
 6. Thenanoparticulate meloxicam composition of claim 1, wherein the at leastone surface stabilizer is polyvinylpyrrolidone.
 7. The nanoparticulatemeloxicam composition of claim 6, wherein the composition consists ofabout 0.5% (w/w) polyvinylpyrrolidone.
 8. The nanoparticulate meloxicamcomposition of claim 1, wherein the at least one surface stabilizer issodium deoxycholate.
 9. The nanoparticulate meloxicam composition ofclaim 8, wherein the composition consists of about 0.25% (w/w) sodiumdeoxycholate as a surface stabilizer.
 10. The nanoparticulate meloxicamcomposition of claim 1, wherein the composition consists of fewer thanabout 1,000 active agent particles that are greater than about 10 μm insize.
 11. The nanoparticulate meloxicam composition of claim 1, whereinthe composition consists of: (a) about 0.5% (w/w) polyvinylpyrrolidoneand about 0.25% (w/w) sodium deoxycholate as the surface stabilizer; and(b) about 5% (w/w) flake-like aggregation reducing agent; wherein theflake-like reducing agent comprises sucrose.
 12. A method ofadministering the injectable, nanoparticulate meloxicam composition ofclaim 1 to a human comprising intravenously, intramuscularly, orsubcutaneously injecting the composition into the human.
 13. A method ofadministering the injectable, nanoparticulate meloxicam composition ofclaim 1 to a human comprising intravenously injecting the compositioninto the human.
 14. The nanoparticulate meloxicam composition of claim1, wherein the composition consists of: (a) about 0.75% (w/w)polyvinylpyrrolidone and about 0.25% (w/w) sodium deoxycholate as thesurface stabilizer; and (b) about 5% (w/w) flake-like aggregationreducing agent; wherein the flake-like reducing agent comprises sucrose.